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Sample records for rapid mutation identification

  1. Rapid identification of HEXA mutations in Tay-Sachs patients.

    Science.gov (United States)

    Giraud, Carole; Dussau, Jeanne; Azouguene, Emilie; Feillet, François; Puech, Jean-Philippe; Caillaud, Catherine

    2010-02-19

    Tay-Sachs disease (TSD) is a recessively inherited neurodegenerative disorder due to mutations in the HEXA gene resulting in a beta-hexosaminidase A (Hex A) deficiency. The purpose of this study was to characterize the molecular abnormalities in patients with infantile or later-onset forms of the disease. The complete sequencing of the 14 exons and flanking regions of the HEXA gene was performed with a unique technical condition in 10 unrelated TSD patients. Eleven mutations were identified, including five splice mutations, one insertion, two deletions and three single-base substitutions. Four mutations were novel: two splice mutations (IVS8+5G>A, IVS2+4delAGTA), one missense mutation in exon 6 (c.621T>G (p.D207E)) and one small deletion (c.1211-1212delTG) in exon 11 resulting in a premature stop codon at residue 429. The c.621T>G missense mutation was found in a patient presenting an infantile form. Its putative role in the pathogenesis of TSD is suspected as residue 207 is highly conserved in human, mouse and rat. Moreover, structural modelling predicted changes likely to affect substrate binding and catalytic activity of the enzyme. The time-saving procedure reported here could be useful for the characterization of Tay-Sachs-causing mutations, in particular in non-Ashkenazi patients mainly exhibiting rare mutations. Copyright (c) 2010 Elsevier Inc. All rights reserved.

  2. Identification of Variant-Specific Functions of PIK3CA by Rapid Phenotyping of Rare Mutations | Office of Cancer Genomics

    Science.gov (United States)

    Large-scale sequencing efforts are uncovering the complexity of cancer genomes, which are composed of causal "driver" mutations that promote tumor progression along with many more pathologically neutral "passenger" events. The majority of mutations, both in known cancer drivers and uncharacterized genes, are generally of low occurrence, highlighting the need to functionally annotate the long tail of infrequent mutations present in heterogeneous cancers.

  3. Rapid identification and recovery of ENU-induced mutations with next-generation sequencing and Paired-End Low-Error analysis.

    Science.gov (United States)

    Pan, Luyuan; Shah, Arish N; Phelps, Ian G; Doherty, Dan; Johnson, Eric A; Moens, Cecilia B

    2015-02-14

    Targeting Induced Local Lesions IN Genomes (TILLING) is a reverse genetics approach to directly identify point mutations in specific genes of interest in genomic DNA from a large chemically mutagenized population. Classical TILLING processes, based on enzymatic detection of mutations in heteroduplex PCR amplicons, are slow and labor intensive. Here we describe a new TILLING strategy in zebrafish using direct next generation sequencing (NGS) of 250 bp amplicons followed by Paired-End Low-Error (PELE) sequence analysis. By pooling a genomic DNA library made from over 9,000 N-ethyl-N-nitrosourea (ENU) mutagenized F1 fish into 32 equal pools of 288 fish, each with a unique Illumina barcode, we reduce the complexity of the template to a level at which we can detect mutations that occur in a single heterozygous fish in the entire library. MiSeq sequencing generates 250 base-pair overlapping paired-end reads, and PELE analysis aligns the overlapping sequences to each other and filters out any imperfect matches, thereby eliminating variants introduced during the sequencing process. We find that this filtering step reduces the number of false positive calls 50-fold without loss of true variant calls. After PELE we were able to validate 61.5% of the mutant calls that occurred at a frequency between 1 mutant call:100 wildtype calls and 1 mutant call:1000 wildtype calls in a pool of 288 fish. We then use high-resolution melt analysis to identify the single heterozygous mutation carrier in the 288-fish pool in which the mutation was identified. Using this NGS-TILLING protocol we validated 28 nonsense or splice site mutations in 20 genes, at a two-fold higher efficiency than using traditional Cel1 screening. We conclude that this approach significantly increases screening efficiency and accuracy at reduced cost and can be applied in a wide range of organisms.

  4. Phenylketonuria mutation analysis in Northern Ireland: A rapid stepwise approach

    Energy Technology Data Exchange (ETDEWEB)

    Zschocke, J.; Graham, C.A.; Nevin, N.C. [Queen`s Univ., Belfast (Australia)] [and others

    1995-12-01

    We present a multistep approach for the rapid analysis of phenylketonuria (PKU) mutations. In the first step, three common mutations and a polymorphic short tandem repeat (STR) system are rapidly analyzed with a fluorescent multiplex assay. In the second step, minihaplotypes combining STR and VNTR data are used to determine rare mutations likely to be present in an investigated patient, which are then confirmed by restriction enzyme analysis. The remaining mutations are analyzed with denaturant gradient-gel electrophoresis and sequencing. The first two steps together identify both mutations in 90%-95% of PKU patients, and results can be obtained within 2 d. We have investigated 121 Northern Irish families with hyperphenylalaninemia, including virtually all patients born since 1972, and have found 34 different mutations on 241 of the 242 mutant alleles. Three mutations (R408W, 165T, and F39L) account for 57.5% of mutations, while 14 mutations occur with a frequency of 1%-6%. The present analysis system is efficient and inexpensive and is particularly well suited to routine mutation analysis in a diagnostic setting. 19 refs., 5 tabs.

  5. Rapid identification of staphylococci by Raman spectroscopy

    Czech Academy of Sciences Publication Activity Database

    Rebrošová, K.; Šiler, Martin; Samek, Ota; Růžička, F.; Bernatová, Silvie; Holá, V.; Ježek, Jan; Zemánek, Pavel; Sokolová, J.; Petráš, P.

    2017-01-01

    Roč. 7, NOV (2017), s. 1-8, č. článku 14846. ISSN 2045-2322 R&D Projects: GA ČR(CZ) GA15-20645S; GA MŠk(CZ) LO1212; GA MŠk ED0017/01/01 Institutional support: RVO:68081731 Keywords : coagulase-negative staphylococci * Raman spectroscopy * rapid identification Subject RIV: BH - Optics, Masers, Lasers OBOR OECD: Optics (including laser optics and quantum optics) Impact factor: 4.259, year: 2016

  6. Rapid lard identification with portable electronic nose

    Science.gov (United States)

    Latief, Marsad; Khorsidtalab, Aida; Saputra, Irwan; Akmeliawati, Rini; Nurashikin, Anis; Jaswir, Irwandi; Witjaksono, Gunawan

    2017-11-01

    Human sensory systems are limited in many different regards, yet they are great sources of inspiration for development of technologies that help humans to overcome their restraints. This paper signifies the capability of our developed electronic nose in rapid lard identification. The developed device, known as E-Nose, mimics human’s olfactory system’s technique to identify a particular substance. Lard is a common pig derivative which is often used as a food additive, emulsion or shortening. It’s also commonly used as an adulterant or as an alternative for cooking oils, margarine and butter. This substance is prohibited to be consumed by Muslims and Orthodox Jews for religious reasons. A portable reliable device with an ability to identify lard rapidly can be convenient to users concerned about lard adulteration. The prototype was examined using K-Nearest Neighbors algorithm (KNN), Support Vector Machine (SVM), Bagged Trees and Simple Tree, and can identify lard with the highest accuracy of 95.6% among three types of fat (lard, chicken and beef) in liquid form over a certain range of temperature using KNN.

  7. Rapid Methods for the Laboratory Identification of Pathogenic Microorganisms.

    Science.gov (United States)

    1982-09-01

    coli Hemophilus influenzae Bacillus anthracis Bacillus circulans Bacillus coagulans Bacillus cereus T Candida albicans Cryptococcus neoformans Legionel...reveree aide If neceeeary and Identify by block number) Lectins: Rapid Identification, Bacillus anthracisjCryptococcus " neoformans. Neisseria...field-type kit for the rapid identification of Bacillus anthracis. We have shown that certain lectins will selectively interact with B. anthracis

  8. Identification of glucose 6 phosphate dehydrogenase mutations by ...

    African Journals Online (AJOL)

    Identification of glucose 6 phosphate dehydrogenase mutations by single strand conformation polymorphism and gene sequencing analysis. ... Subject: Six DNA samples from Turkish males confirmed to have G-6-PD deficiency where available for the study. Results: One subject was found to have an abnormal mobility shift ...

  9. Rapid BRAF mutation tests in patients with advanced melanoma : Comparison of immunohistochemistry, Droplet Digital PCR, and the Idylla Mutation Platform

    NARCIS (Netherlands)

    Bisschop, Cornelis; Ter Elst, Arja; Bosman, Lisette J; Platteel, Inge; Jalving, Mathilde; van den Berg, Anke; Diepstra, Arjan; van Hemel, Bettien; Diercks, Gilles F H; Hospers, Geke A P; Schuuring, Ed

    BRAF mutational testing has become a common practice in the diagnostic process of patients with advanced melanoma. Although time-consuming, DNA sequencing techniques are the current gold standard for mutational testing. However, in certain clinical situations, a rapid test result is required. In

  10. Rapid detection of RB1 recurrent mutations in retinoblastoma by ...

    Indian Academy of Sciences (India)

    In about half of the patients, one mutation is inherited via the germinal cells, while in the .... mutational hot spots in the RB1 gene, making genetic testing complex and challenging ... by direct sequencing. High normal background in sequenc-.

  11. Rapid Identification of Bacterial Virulence Factors

    Science.gov (United States)

    2014-04-15

    protein sorting and transport. F/’/wyi-deletion mutants had decreased invasiveness of HeLa cells when compared to their parental strain, and it has...mileux. Bacteria with intracellular life styles and have reductive genomes often have many different ABC transporters. This is certainly the case in...34 Microbiology 151:2975-2986. Newman , R.M., P. Salunkhe, A. Godzik, J.C. Reed. 2006. Identification and Characterization of a Novel Bacterial

  12. A parylene-based dual channel microelectrophoresis system for rapid mutation detection via heteroduplex analysis

    NARCIS (Netherlands)

    Sukas, S.; Erson, Ayse Elif; Sert, Cuneyt; Kulah, Haluk

    2008-01-01

    A new dual channel micro-electrophoresis system for rapid mutation detection based on heteroduplex analysis was designed and implemented. Mutation detection was successfully achieved in a total separation length of 250 μm in less than 3 min for a 590 bp DNA sample harboring a 3 bp mutation causing

  13. The rapid identification for the unaware radioactive material

    International Nuclear Information System (INIS)

    Jin Yuren; Cheng Zhiwei; Xu Hui; Wang Jiang; Han Xiaoyuan; Long Bin

    2010-01-01

    The unaware radioactive material(URM) appeared in the society may induce serious deterministic effect, even result in havoc and instability of the society. The rapid and accurate identification for URM is the premise for its reasonable treatment. In this paper, an identification procedure for URM was developed and which was successfully implemented in the identification of an URM. The In-situ HPGe gamma spectrometry etc was employed for the rapid preliminary identification, and the laboratory HPGe gamma spectrometry and ICP-MS as well as the density measurement were used for its final identification. One unaware radioactive material was assayed, and the results indicate that it is a kind of high pure depleted uranium metal with the 235 U/ 238 U atomic ratio of 0.454%. (authors)

  14. Rapid identification of DNA-binding proteins by mass spectrometry

    DEFF Research Database (Denmark)

    Nordhoff, E.; Korgsdam, A.-M.; Jørgensen, H.F.

    1999-01-01

    We report a protocol for the rapid identification of DNA-binding proteins. Immobilized DNA probes harboring a specific sequence motif are incubated with cell or nuclear extract. Proteins are analyzed directly off the solid support by matrix-assisted laser desorption/ionization time-of-flight mass...... was validated by the identification of known prokaryotic and eukaryotic DNA-binding proteins, and its use provided evidence that poly(ADP-ribose) polymerase exhibits DNA sequence-specific binding to DNA....

  15. Identification of HIV Mutation as Diagnostic Biomarker through Next Generation Sequencing.

    Science.gov (United States)

    Shaw, Wen Hui; Lin, Qianqian; Muhammad, Zikry Zhiwei Bin Roslee; Lee, Jia Jun; Khong, Wei Xin; Ng, Oon Tek; Tan, Eng Lee; Li, Peng

    2016-07-01

    Current clinical detection of Human immunodeficiency virus 1 (HIV-1) is used to target viral genes and proteins. However, the immunoassay, such as viral culture or Polymerase Chain Reaction (PCR), lacks accuracy in the diagnosis, as these conventional assays rely on the stable genome and HIV-1 is a highly-mutated virus. Next generation sequencing (NGS) promises to be transformative for the practice of infectious disease, and the rapidly reducing cost and processing time mean that this will become a feasible technology in diagnostic and research laboratories in the near future. The technology offers the superior sensitivity to detect the pathogenic viruses, including unknown and unexpected strains. To leverage the NGS technology in order to improve current HIV-1 diagnosis and genotyping methods. Ten blood samples were collected from HIV-1 infected patients which were diagnosed by RT PCR at Singapore Communicable Disease Centre, Tan Tock Seng Hospital from October 2014 to March 2015. Viral RNAs were extracted from blood plasma and reversed into cDNA. The HIV-1 cDNA samples were cleaned up using a PCR purification kit and the sequencing library was prepared and identified through MiSeq. Two common mutations were observed in all ten samples. The common mutations were identified at genome locations 1908 and 2104 as missense and silent mutations respectively, conferring S37N and S3S found on aspartic protease and reverse transcriptase subunits. The common mutations identified in this study were not previously reported, therefore suggesting the potential for them to be used for identification of viral infection, disease transmission and drug resistance. This was especially the case for, missense mutation S37N which could cause an amino acid change in viral proteases thus reducing the binding affinity of some protease inhibitors. Thus, the unique common mutations identified in this study could be used as diagnostic biomarkers to indicate the origin of infection as being

  16. Deletion Mutagenesis and Identification of Causative Mutations in Maize.

    Science.gov (United States)

    Jia, Shangang; Li, Aixia; Zhang, Chi; Holding, David

    2018-01-01

    We describe a method for gamma-irradiation of mature maize seeds to generate mutants with opaque endosperm and reduced kernel fill phenotypes. We also describe methods for mapping mutants and identifying causal gene mutations. Using this method, a population of 1788M2 families and 47 Mo17 × F2s showing stable, segregating, and viable kernel phenotypes was developed. For molecular characterization of the mutants, we utilized a novel functional genomics platform that combines separate Bulked Segregant RNA and exome sequencing data sets (BSREx-seq) to map causative mutations and identify candidate genes within mapping intervals. We also describe the use of exome capture sequencing of F2 mutant and normal pools to perform mapping and candidate gene identification without the need for separate RNA-seq (BSEx-seq). To exemplify the utility of the deletion mutants for functional genomics and provide proof-of-concept for the bioinformatics platform, we summarize the identification of the causative deletion in two mutants. Mutant 937, which was characterized by BSREx-seq, harbors a 6203-bp in-frame deletion covering six exons within the Opaque-1 gene on chromosome 4. Preliminary investigation of opaque mutant 1486 with BSEx-seq shows a tight mapping interval and associated deletion on chromosome 10.

  17. Development of rapid phenotypic system for the identification

    Indian Academy of Sciences (India)

    Rapid and accurate identification of bacterial pathogens is a fundamental goal of clinical microbiology. The diagnosis and surveillance of diseases is dependent, to a great extent, on laboratory services, which cannot function without effective reliable reagents and diagnostics. Despite the advancement in microbiology ...

  18. High resolution melting analysis: a rapid and accurate method to detect CALR mutations.

    Directory of Open Access Journals (Sweden)

    Cristina Bilbao-Sieyro

    Full Text Available The recent discovery of CALR mutations in essential thrombocythemia (ET and primary myelofibrosis (PMF patients without JAK2/MPL mutations has emerged as a relevant finding for the molecular diagnosis of these myeloproliferative neoplasms (MPN. We tested the feasibility of high-resolution melting (HRM as a screening method for rapid detection of CALR mutations.CALR was studied in wild-type JAK2/MPL patients including 34 ET, 21 persistent thrombocytosis suggestive of MPN and 98 suspected secondary thrombocytosis. CALR mutation analysis was performed through HRM and Sanger sequencing. We compared clinical features of CALR-mutated versus 45 JAK2/MPL-mutated subjects in ET.Nineteen samples showed distinct HRM patterns from wild-type. Of them, 18 were mutations and one a polymorphism as confirmed by direct sequencing. CALR mutations were present in 44% of ET (15/34, 14% of persistent thrombocytosis suggestive of MPN (3/21 and none of the secondary thrombocytosis (0/98. Of the 18 mutants, 9 were 52 bp deletions, 8 were 5 bp insertions and other was a complex mutation with insertion/deletion. No mutations were found after sequencing analysis of 45 samples displaying wild-type HRM curves. HRM technique was reproducible, no false positive or negative were detected and the limit of detection was of 3%.This study establishes a sensitive, reliable and rapid HRM method to screen for the presence of CALR mutations.

  19. Two rapid pigmentation tests for identification of Cryptococcus neoformans.

    Science.gov (United States)

    Kaufmann, C S; Merz, W G

    1982-01-01

    Two tests were developed for the rapid identification of Cryptococcus neoformans based on pigment produced by the organism's phenoloxidase activity. Caffeic acid was incorporated into cornmeal agar, a medium used routinely for yeast identification. When tested on this medium, only C. neoformans isolates produced brown pigment. All other yeasts maintained their normal morphology and did not produce the reaction product. A non-medium-based test was developed for same-day identification of C. neoformans isolates. Paper strips saturated with a buffered L-beta-3,4-dihydroxyphenylalanine-ferric citrate solution were inoculated with isolates and incubated at 37 degrees C. Pigment production occurred only with C. neoformans isolates, many within 60 to 90 min. All other yeasts remained negative. PMID:7040452

  20. NMD Microarray Analysis for Rapid Genome-Wide Screen of Mutated Genes in Cancer

    Directory of Open Access Journals (Sweden)

    Maija Wolf

    2005-01-01

    Full Text Available Gene mutations play a critical role in cancer development and progression, and their identification offers possibilities for accurate diagnostics and therapeutic targeting. Finding genes undergoing mutations is challenging and slow, even in the post-genomic era. A new approach was recently developed by Noensie and Dietz to prioritize and focus the search, making use of nonsense-mediated mRNA decay (NMD inhibition and microarray analysis (NMD microarrays in the identification of transcripts containing nonsense mutations. We combined NMD microarrays with array-based CGH (comparative genomic hybridization in order to identify inactivation of tumor suppressor genes in cancer. Such a “mutatomics” screening of prostate cancer cell lines led to the identification of inactivating mutations in the EPHB2 gene. Up to 8% of metastatic uncultured prostate cancers also showed mutations of this gene whose loss of function may confer loss of tissue architecture. NMD microarray analysis could turn out to be a powerful research method to identify novel mutated genes in cancer cell lines, providing targets that could then be further investigated for their clinical relevance and therapeutic potential.

  1. Continuous-Flow Detector for Rapid Pathogen Identification

    Energy Technology Data Exchange (ETDEWEB)

    Barrett, Louise M. [Sandia National Lab. (SNL-CA), Livermore, CA (United States). Microfluidics; Skulan, Andrew J. [Sandia National Lab. (SNL-CA), Livermore, CA (United States). Microfluidics; Singh, Anup K. [Sandia National Lab. (SNL-CA), Livermore, CA (United States). Microfluidics; Cummings, Eric B. [Sandia National Lab. (SNL-CA), Livermore, CA (United States). Microfluidics; Fiechtner, Gregory J. [Sandia National Lab. (SNL-CA), Livermore, CA (United States). Microfluidics

    2006-09-01

    This report describes the continued development of a low-power, portable detector for the rapid identification of pathogens such as B. anthracis and smallpox. Based on our successful demonstration of the continuous filter/concentrator inlet, we believe strongly that the inlet section will enable differentiation between viable and non-viable populations, between types of cells, and between pathogens and background contamination. Selective, continuous focusing of particles in a microstream enables highly selective and sensitive identification using fluorescently labeled antibodies and other receptors such as peptides, aptamers, or small ligands to minimize false positives. Processes such as mixing and lysing will also benefit from the highly localized particle streams. The concentrator is based on faceted prisms to contract microfluidic flows while maintaining uniform flowfields. The resulting interfaces, capable of high throughput, serve as high-, low-, and band-pass filters to direct selected bioparticles to a rapid, affinity-based detection system. The proposed device is superior to existing array-based detectors as antibody-pathogen binding can be accomplished in seconds rather than tens of minutes or even hours. The system is being designed to interface with aerosol collectors under development by the National Laboratories or commercial systems. The focused stream is designed to be interrogated using diode lasers to differentiate pathogens by light scattering. Identification of particles is done using fluorescently labeled antibodies to tag the particles, followed by multiplexed laser-induced fluorescence (LIF) detection (achieved by labeling each antibody with a different dye).

  2. Pyrosequencing-Based Assays for Rapid Detection of HER2 and HER3 Mutations in Clinical Samples Uncover an E332E Mutation Affecting HER3 in Retroperitoneal Leiomyosarcoma

    Directory of Open Access Journals (Sweden)

    Paula González-Alonso

    2015-08-01

    Full Text Available Mutations in Human Epidermal Growth Factor Receptors (HER are associated with poor prognosis of several types of solid tumors. Although HER-mutation detection methods are currently available, such as Next-Generation Sequencing (NGS, alternative pyrosequencing allow the rapid characterization of specific mutations. We developed specific PCR-based pyrosequencing assays for identification of most prevalent HER2 and HER3 mutations, including S310F/Y, R678Q, L755M/P/S/W, V777A/L/M, 774-776 insertion, and V842I mutations in HER2, as well as M91I, V104M/L, D297N/V/Y, and E332E/K mutations in HER3. We tested 85 Formalin Fixed and Paraffin Embbeded (FFPE samples and we detected three HER2-V842I mutations in colorectal carcinoma (CRC, ovarian carcinoma, and pancreatic carcinoma patients, respectively, and a HER2-L755M mutation in a CRC specimen. We also determined the presence of a HER3-E332K mutation in an urothelial carcinoma sample, and two HER3-D297Y mutations, in both gastric adenocarcinoma and CRC specimens. The D297Y mutation was previously detected in breast and gastric tumors, but not in CRC. Moreover, we found a not-previously-described HER3-E332E synonymous mutation in a retroperitoneal leiomyosarcoma patient. The pyrosequencing assays presented here allow the detection and characterization of specific HER2 and HER3 mutations. These pyrosequencing assays might be implemented in routine diagnosis for molecular characterization of HER2/HER3 receptors as an alternative to complex NGS approaches.

  3. Rapid Molecular Identification of Human Taeniid Cestodes by Pyrosequencing Approach

    Science.gov (United States)

    Thanchomnang, Tongjit; Tantrawatpan, Chairat; Intapan, Pewpan M.; Sanpool, Oranuch; Janwan, Penchom; Lulitanond, Viraphong; Tourtip, Somjintana; Yamasaki, Hiroshi; Maleewong, Wanchai

    2014-01-01

    Taenia saginata, T. solium, and T. asiatica are causative agents of taeniasis in humans. The difficulty of morphological identification of human taeniids can lead to misdiagnosis or confusion. To overcome this problem, several molecular methods have been developed, but use of these tends to be time-consuming. Here, a rapid and high-throughput pyrosequencing approach was developed for the identification of three human taeniids originating from various countries. Primers targeting the mitochondrial cytochrome c oxidase subunit 1 (cox1) gene of the three Taenia species were designed. Variations in a 26-nucleotide target region were used for identification. The reproducibility and accuracy of the pyrosequencing technology was confirmed by Sanger sequencing. This technique will be a valuable tool to distinguish between sympatric human taeniids that occur in Thailand, Asia and Pacific countries. This method could potentially be used for the molecular identification of the taeniid species that might be associated with suspicious cysts and lesions, or cyst residues in humans or livestock at the slaughterhouse. PMID:24945530

  4. Rapid molecular identification of human taeniid cestodes by pyrosequencing approach.

    Directory of Open Access Journals (Sweden)

    Tongjit Thanchomnang

    Full Text Available Taenia saginata, T. solium, and T. asiatica are causative agents of taeniasis in humans. The difficulty of morphological identification of human taeniids can lead to misdiagnosis or confusion. To overcome this problem, several molecular methods have been developed, but use of these tends to be time-consuming. Here, a rapid and high-throughput pyrosequencing approach was developed for the identification of three human taeniids originating from various countries. Primers targeting the mitochondrial cytochrome c oxidase subunit 1 (cox1 gene of the three Taenia species were designed. Variations in a 26-nucleotide target region were used for identification. The reproducibility and accuracy of the pyrosequencing technology was confirmed by Sanger sequencing. This technique will be a valuable tool to distinguish between sympatric human taeniids that occur in Thailand, Asia and Pacific countries. This method could potentially be used for the molecular identification of the taeniid species that might be associated with suspicious cysts and lesions, or cyst residues in humans or livestock at the slaughterhouse.

  5. Rapid molecular identification and characteristics of Lactobacillus strains.

    Science.gov (United States)

    Markiewicz, L H; Biedrzycka, E; Wasilewska, E; Bielecka, M

    2010-09-01

    Eleven type strains and 24 Lactobacillus isolates, preliminarily classified to the species due to phenotypic features, were investigated. Standard methods of identification with species-specific PCRs and typing with PFGE (with ApaI, NotI and SmaI restriction enzymes) allowed us to distinguish 16 unique strains belonging to 5 species (L. acidophilus, L. delbrueckii ssp. bulgaricus, L. plantarum, L. rhamnosus, L. salivarius). Alternative approach with 16S-23S rDNA ARDRA identification (with merely two restrictases, BsuRI and TaqI) and PCR-based typing (RAPD with two random- and rep-PCR with (GTG)(5) primers) showed to be more discriminative, i.e. 21 unique strains were classified in the same species as above. As a result, 7 out of 24 phenotypically species-assigned isolates were reclassified. The alternative procedure of rapid identification and typing of Lactobacillus isolates appeared to be equally effective and shortened from 1 week to 2-3 d (in comparison to the standard methods).

  6. High-resolution melting curve analysis for rapid detection of mutations in a Medaka TILLING library

    Directory of Open Access Journals (Sweden)

    Deguchi Tomonori

    2010-09-01

    Full Text Available Abstract Background During the last two decades, DNA sequencing has led to the identification of numerous genes in key species; however, in most cases, their functions are still unknown. In this situation, reverse genetics is the most suitable method to assign function to a gene. TILLING (Targeting Induced Local Lesions IN Genomes is a reverse-genetic strategy that combines random chemical mutagenesis with high-throughput discovery of the induced mutations in target genes. The method has been applied to a variety of plant and animal species. Screening of the induced mutations is the most important step in TILLING. Currently, direct sequencing or nuclease-mediated screening of heteroduplexes is widely used for detection of mutations in TILLING. Both methods are useful, but the costs are substantial and turnaround times are relatively long. Thus, there is a need for an alternative method that is of higher throughput and more cost effective. Results In this study, we developed a high resolution melting (HRM assay and evaluated its effectiveness for screening ENU-induced mutations in a medaka TILLING library. We had previously screened mutations in the p53 gene by direct sequencing. Therefore, we first tested the efficiency of the HRM assay by screening mutations in p53, which indicated that the HRM assay is as useful as direct sequencing. Next, we screened mutations in the atr and atm genes with the HRM assay. Nonsense mutations were identified in each gene, and the phenotypes of these nonsense mutants confirmed their loss-of-function nature. Conclusions These results demonstrate that the HRM assay is useful for screening mutations in TILLING. Furthermore, the phenotype of the obtained mutants indicates that medaka is an excellent animal model for investigating genome stability and gene function, especially when combined with TILLING.

  7. Validation of high-resolution DNA melting analysis for mutation scanning of the CDKL5 gene: identification of novel mutations.

    Science.gov (United States)

    Raymond, Laure; Diebold, Bertrand; Leroux, Céline; Maurey, Hélène; Drouin-Garraud, Valérie; Delahaye, Andre; Dulac, Olivier; Metreau, Julia; Melikishvili, Gia; Toutain, Annick; Rivier, François; Bahi-Buisson, Nadia; Bienvenu, Thierry

    2013-01-01

    Mutations in the cyclin-dependent kinase-like 5 gene (CDKL5) have been predominantly described in epileptic encephalopathies of female, including infantile spasms with Rett-like features. Up to now, detection of mutations in this gene was made by laborious, expensive and/or time consuming methods. Here, we decided to validate high-resolution melting analysis (HRMA) for mutation scanning of the CDKL5 gene. Firstly, using a large DNA bank consisting to 34 samples carrying different mutations and polymorphisms, we validated our analytical conditions to analyse the different exons and flanking intronic sequences of the CDKL5 gene by HRMA. Secondly, we screened CDKL5 by both HRMA and denaturing high performance liquid chromatography (dHPLC) in a cohort of 135 patients with early-onset seizures. Our results showed that point mutations and small insertions and deletions can be reliably detected by HRMA. Compared to dHPLC, HRMA profiles are more discriminated, thereby decreasing unnecessary sequencing. In this study, we identified eleven novel sequence variations including four pathogenic mutations (2.96% prevalence). HRMA appears cost-effective, easy to set up, highly sensitive, non-toxic and rapid for mutation screening, ideally suited for large genes with heterogeneous mutations located along the whole coding sequence, such as the CDKL5 gene. Copyright © 2012 Elsevier B.V. All rights reserved.

  8. Passive IFF: Autonomous Nonintrusive Rapid Identification of Friendly Assets

    Science.gov (United States)

    Moynihan, Philip; Steenburg, Robert Van; Chao, Tien-Hsin

    2004-01-01

    A proposed optoelectronic instrument would identify targets rapidly, without need to radiate an interrogating signal, apply identifying marks to the targets, or equip the targets with transponders. The instrument was conceived as an identification, friend or foe (IFF) system in a battlefield setting, where it would be part of a targeting system for weapons, by providing rapid identification for aimed weapons to help in deciding whether and when to trigger them. The instrument could also be adapted to law-enforcement and industrial applications in which it is necessary to rapidly identify objects in view. The instrument would comprise mainly an optical correlator and a neural processor (see figure). The inherent parallel-processing speed and capability of the optical correlator would be exploited to obtain rapid identification of a set of probable targets within a scene of interest and to define regions within the scene for the neural processor to analyze. The neural processor would then concentrate on each region selected by the optical correlator in an effort to identify the target. Depending on whether or not a target was recognized by comparison of its image data with data in an internal database on which the neural processor was trained, the processor would generate an identifying signal (typically, friend or foe ). The time taken for this identification process would be less than the time needed by a human or robotic gunner to acquire a view of, and aim at, a target. An optical correlator that has been under development for several years and that has been demonstrated to be capable of tracking a cruise missile might be considered a prototype of the optical correlator in the proposed IFF instrument. This optical correlator features a 512-by-512-pixel input image frame and operates at an input frame rate of 60 Hz. It includes a spatial light modulator (SLM) for video-to-optical image conversion, a pair of precise lenses to effect Fourier transforms, a filter SLM

  9. Identification of a novel CLRN1 gene mutation in Usher syndrome type 3: two case reports.

    Science.gov (United States)

    Yoshimura, Hidekane; Oshikawa, Chie; Nakayama, Jun; Moteki, Hideaki; Usami, Shin-Ichi

    2015-05-01

    This study examines the CLRN1 gene mutation analysis in Japanese patients who were diagnosed with Usher syndrome type 3 (USH3) on the basis of clinical findings. Genetic analysis using massively parallel DNA sequencing (MPS) was conducted to search for 9 causative USH genes in 2 USH3 patients. We identified the novel pathogenic mutation in the CLRN1 gene in 2 patients. The missense mutation was confirmed by functional prediction software and segregation analysis. Both patients were diagnosed as having USH3 caused by the CLRN1 gene mutation. This is the first report of USH3 with a CLRN1 gene mutation in Asian populations. Validating the presence of clinical findings is imperative for properly differentiating among USH subtypes. In addition, mutation screening using MPS enables the identification of causative mutations in USH. The clinical diagnosis of this phenotypically variable disease can then be confirmed. © The Author(s) 2015.

  10. Identification of Constrained Cancer Driver Genes Based on Mutation Timing

    Science.gov (United States)

    Sakoparnig, Thomas; Fried, Patrick; Beerenwinkel, Niko

    2015-01-01

    Cancer drivers are genomic alterations that provide cells containing them with a selective advantage over their local competitors, whereas neutral passengers do not change the somatic fitness of cells. Cancer-driving mutations are usually discriminated from passenger mutations by their higher degree of recurrence in tumor samples. However, there is increasing evidence that many additional driver mutations may exist that occur at very low frequencies among tumors. This observation has prompted alternative methods for driver detection, including finding groups of mutually exclusive mutations and incorporating prior biological knowledge about gene function or network structure. Dependencies among drivers due to epistatic interactions can also result in low mutation frequencies, but this effect has been ignored in driver detection so far. Here, we present a new computational approach for identifying genomic alterations that occur at low frequencies because they depend on other events. Unlike passengers, these constrained mutations display punctuated patterns of occurrence in time. We test this driver–passenger discrimination approach based on mutation timing in extensive simulation studies, and we apply it to cross-sectional copy number alteration (CNA) data from ovarian cancer, CNA and single-nucleotide variant (SNV) data from breast tumors and SNV data from colorectal cancer. Among the top ranked predicted drivers, we find low-frequency genes that have already been shown to be involved in carcinogenesis, as well as many new candidate drivers. The mutation timing approach is orthogonal and complementary to existing driver prediction methods. It will help identifying from cancer genome data the alterations that drive tumor progression. PMID:25569148

  11. Identification of Germline Genetic Mutations in Pancreatic Cancer Patients

    Science.gov (United States)

    Salo-Mullen, Erin E.; O’Reilly, Eileen; Kelsen, David; Ashraf, Asad M.; Lowery, Maeve; Yu, Kenneth; Reidy, Diane; Epstein, Andrew S.; Lincoln, Anne; Saldia, Amethyst; Jacobs, Lauren M.; Rau-Murthy, Rohini; Zhang, Liying; Kurtz, Robert; Saltz, Leonard; Offit, Kenneth; Robson, Mark; Stadler, Zsofia K.

    2016-01-01

    Background Pancreatic adenocarcinoma (PAC) is part of several cancer predisposition syndromes; however, indications for genetic counseling/testing are not well-defined. We sought to determine mutation prevalence and characteristics that predict for inherited predisposition to PAC. Methods We identified 175 consecutive PAC patients who underwent clinical genetics assessment at Memorial Sloan Kettering between 2011–2014. Clinical data, family history, and germline results were evaluated. Results Among 159 PAC patients who pursued genetic testing, 24 pathogenic mutations were identified (15.1%; 95%CI, 9.5%–20.7%), including BRCA2(n=13), BRCA1(n=4), p16(n=2), PALB2(n=1), and Lynch syndrome(n=4). BRCA1/BRCA2 prevalence was 13.7% in Ashkenazi Jewish(AJ) (n=95) and 7.1% in non-AJ(n=56) patients. In AJ patients with strong, weak, or absent family history of BRCA-associated cancers, mutation prevalence was 16.7%, 15.8%, and 7.4%, respectively. Mean age at diagnosis in all mutation carriers was 58.5y(range 45–75y) compared to 64y(range 27–87y) in non-mutation carriers(P=0.02). Although BRCA2 was the most common mutation identified, no patients with early-onset PAC(≤50y) harbored a BRCA2 mutation and the mean age at diagnosis in BRCA2 carriers was equivalent to non-mutation carriers(P=0.34). Mutation prevalence in early-onset patients(n=21) was 28.6%, including BRCA1(n=2), p16(n=2), MSH2(n=1) and MLH1(n=1). Conclusion Mutations in BRCA2 account for over 50% of PAC patients with an identified susceptibility syndrome. AJ patients had high BRCA1/BRCA2 prevalence regardless of personal/family history, suggesting that ancestry alone indicates a need for genetic evaluation. With the exception of BRCA2-associated PAC, inherited predisposition to PAC is associated with earlier age at PAC diagnosis suggesting that this subset of patients may also represent a population warranting further evaluation. PMID:26440929

  12. Clinicopathological Correlates in a PRNP P102L Mutation Carrier with Rapidly Progressing Parkinsonism-dystonia

    Science.gov (United States)

    Umeh, Chizoba C.; Kalakoti, Piyush; Greenberg, Michael K; Notari, Silvio; Cohen, Yvonne; Gambetti, Pierluigi; Oblak, Adrian L.; Ghetti, Bernardino; Mari, Zoltan

    2015-01-01

    Parkinsonism-dystonia is rare in carriers of PRNP P102L mutation. Severity and distribution of prion protein (PrP) deposition may influence the clinical presentation. We present such clinic-pathological correlation in a 56-year-old male with a PRNP P102L mutation associated with a phenotype characterized by rapidly progressing parkinsonism-dystonia. The patient was studied clinically (videotaped exams, brain MRIs); molecular genetically (gene sequence analysis); and neuropathologically (histology, immunohistochemistry) during his 7-month disease course. The patient had parkinsonism, apraxia, aphasia, and dystonia, which progressed rapidly. Molecular genetic analysis revealed PRNP P102L mutation carrier status. Brain MRIs revealed progressive global volume loss and T2/FLAIR hyperintensity in neocortex and basal ganglia. Postmortem examination showed neuronal loss, gliosis, spongiform changes, and PrP deposition in the striatum. PrP immunohistochemistry revealed widespread severe PrP deposition in the thalamus and cerebellar cortex. Based on the neuropathological and molecular-genetic analysis, the rapidly progressing parkinsonism-dystonia correlated with nigrostriatal, thalamic, and cerebellar pathology. PMID:27617269

  13. Identification of MPL R102P Mutation in Hereditary Thrombocytosis.

    Science.gov (United States)

    Bellanné-Chantelot, Christine; Mosca, Matthieu; Marty, Caroline; Favier, Rémi; Vainchenker, William; Plo, Isabelle

    2017-01-01

    The molecular basis of hereditary thrombocytosis is germline mutations affecting the thrombopoietin (TPO)/TPO receptor (MPL)/JAK2 signaling axis. Here, we report one family presenting two cases with a mild thrombocytosis. By sequencing JAK2 and MPL coding exons, we identified a germline MPL R102P heterozygous mutation in the proband and his daughter. Concomitantly, we detected high TPO levels in the serum of these two patients. The mutation was not found in three other unaffected cases from the family except in another proband's daughter who did not present thrombocytosis but had a high TPO level. The MPL R102P mutation was first described in congenital amegakaryocytic thrombocytopenia in a homozygous state with a loss-of-function activity. It was previously shown that MPL R102P was blocked in the endoplasmic reticulum without being able to translocate to the plasma membrane. Thus, this case report identifies for the first time that MPL R102P mutation can differently impact megakaryopoiesis: thrombocytosis or thrombocytopenia depending on the presence of the heterozygous or homozygous state, respectively. The paradoxical effect associated with heterozygous MPL R102P may be due to subnormal cell-surface expression of wild-type MPL in platelets inducing a defective TPO clearance. As a consequence, increased TPO levels may activate megakaryocyte progenitors that express a lower, but still sufficient level of MPL for the induction of proliferation.

  14. Identification of MPL R102P Mutation in Hereditary Thrombocytosis

    Directory of Open Access Journals (Sweden)

    Christine Bellanné-Chantelot

    2017-09-01

    Full Text Available The molecular basis of hereditary thrombocytosis is germline mutations affecting the thrombopoietin (TPO/TPO receptor (MPL/JAK2 signaling axis. Here, we report one family presenting two cases with a mild thrombocytosis. By sequencing JAK2 and MPL coding exons, we identified a germline MPL R102P heterozygous mutation in the proband and his daughter. Concomitantly, we detected high TPO levels in the serum of these two patients. The mutation was not found in three other unaffected cases from the family except in another proband’s daughter who did not present thrombocytosis but had a high TPO level. The MPL R102P mutation was first described in congenital amegakaryocytic thrombocytopenia in a homozygous state with a loss-of-function activity. It was previously shown that MPL R102P was blocked in the endoplasmic reticulum without being able to translocate to the plasma membrane. Thus, this case report identifies for the first time that MPL R102P mutation can differently impact megakaryopoiesis: thrombocytosis or thrombocytopenia depending on the presence of the heterozygous or homozygous state, respectively. The paradoxical effect associated with heterozygous MPL R102P may be due to subnormal cell-surface expression of wild-type MPL in platelets inducing a defective TPO clearance. As a consequence, increased TPO levels may activate megakaryocyte progenitors that express a lower, but still sufficient level of MPL for the induction of proliferation.

  15. Hereditary non-polyposis colorectal cancer : Identification of mutation carriers and assessing pathogenicity of mutations

    NARCIS (Netherlands)

    Niessen, RC; Sijmons, RH; Berends, MJW; Ou, J; Hofstra, RNW; Kleibeuker, JH

    2004-01-01

    Hereditary non-polyposis colorectal cancer (HNPCC), also referred to as Lynch syndrome, is an autosomal dominantly inherited disorder that is characterized by susceptibility to colorectal cancer and extracolonic malignancies, in particular endometrial cancer. HNPCC is caused by pathogenic mutations

  16. Rapid Identification and Verification of Indirubin-Containing Medicinal Plants.

    Science.gov (United States)

    Hu, Zhigang; Tu, Yuan; Xia, Ye; Cheng, Peipei; Sun, Wei; Shi, Yuhua; Guo, Licheng; He, Haibo; Xiong, Chao; Chen, Shilin; Zhang, Xiuqiao

    2015-01-01

    Indirubin, one of the key components of medicinal plants including Isatis tinctoria, Polygonum tinctorium, and Strobilanthes cusia, possesses great medicinal efficacy in the treatment of chronic myelocytic leukemia (CML). Due to misidentification and similar name, materials containing indirubin and their close relatives frequently fall prey to adulteration. In this study, we selected an internal transcribed spacer 2 (ITS2) for distinguishing these indirubin-containing species from five of their usual adulterants, after assessing identification efficiency of matK, rbcL, psbA-trnH, and ITS2 among these species. The results of genetic distances and neighbor-joining (NJ) phylogenetic tree indicated that ITS2 region is a powerful DNA barcode to accurately identify these indirubin-containing species and discriminate them from their adulterants. Additionally, high performance liquid chromatography (HPLC) was used to verify indirubin in different organs of the above species. The results showed that indirubin had been detected in the leaves of Is. tinctoria, P. tinctorium, S. cusia, and Indigo Naturalis (made from their mixture), but not in their roots, or in the leaves of their adulterants. Therefore, this study provides a novel and rapid method to identify and verify indirubin-containing medicinal plants for effective natural treatment of CML.

  17. Rapid Identification and Verification of Indirubin-Containing Medicinal Plants

    Directory of Open Access Journals (Sweden)

    Zhigang Hu

    2015-01-01

    Full Text Available Indirubin, one of the key components of medicinal plants including Isatis tinctoria, Polygonum tinctorium, and Strobilanthes cusia, possesses great medicinal efficacy in the treatment of chronic myelocytic leukemia (CML. Due to misidentification and similar name, materials containing indirubin and their close relatives frequently fall prey to adulteration. In this study, we selected an internal transcribed spacer 2 (ITS2 for distinguishing these indirubin-containing species from five of their usual adulterants, after assessing identification efficiency of matK, rbcL, psbA-trnH, and ITS2 among these species. The results of genetic distances and neighbor-joining (NJ phylogenetic tree indicated that ITS2 region is a powerful DNA barcode to accurately identify these indirubin-containing species and discriminate them from their adulterants. Additionally, high performance liquid chromatography (HPLC was used to verify indirubin in different organs of the above species. The results showed that indirubin had been detected in the leaves of Is. tinctoria, P. tinctorium, S. cusia, and Indigo Naturalis (made from their mixture, but not in their roots, or in the leaves of their adulterants. Therefore, this study provides a novel and rapid method to identify and verify indirubin-containing medicinal plants for effective natural treatment of CML.

  18. Efficiency of Database Search for Identification of Mutated and Modified Proteins via Mass Spectrometry

    OpenAIRE

    Pevzner, Pavel A.; Mulyukov, Zufar; Dancik, Vlado; Tang, Chris L

    2001-01-01

    Although protein identification by matching tandem mass spectra (MS/MS) against protein databases is a widespread tool in mass spectrometry, the question about reliability of such searches remains open. Absence of rigorous significance scores in MS/MS database search makes it difficult to discard random database hits and may lead to erroneous protein identification, particularly in the case of mutated or post-translationally modified peptides. This problem is especially important for high-thr...

  19. Identification of a Novel Mutation in a Pseudohypoparathyroidism Family

    Directory of Open Access Journals (Sweden)

    Zhi-Min Miao

    2011-01-01

    Full Text Available Pseudohypoparathyroidism type Ia (PHP Ia is defined as a series of disorders characterized by multihormone resistance in end-organs and Albright hereditary osteodystrophy (AHO phenotype. PHP Ia is caused by heterozygous inactivating mutations in GNAS, which encodes the stimulatory G-protein alpha subunit (Gsa. A patient with typical clinical manifestations of pseudohypoparathyroidism (PHP (round face, short stature, centripetal obesity, brachydactyly, and multi-hormone resistance: parathyroid hormone (PTH, thyroid-stimulating hormone (TSH, and gonadotropins presented at our center. The sequence of the GNAS gene from the patient and her families revealed a novel missense mutation (Y318H in the proband and her mother. An in vitro Gsa functional study showed that Gsa function was significantly impaired. These results stress the importance of GNAS gene investigation.

  20. Neurogenomics and the role of a large mutational target on rapid behavioral change.

    Science.gov (United States)

    Stanley, Craig E; Kulathinal, Rob J

    2016-11-08

    Behavior, while complex and dynamic, is among the most diverse, derived, and rapidly evolving traits in animals. The highly labile nature of heritable behavioral change is observed in such evolutionary phenomena as the emergence of converged behaviors in domesticated animals, the rapid evolution of preferences, and the routine development of ethological isolation between diverging populations and species. In fact, it is believed that nervous system development and its potential to evolve a seemingly infinite array of behavioral innovations played a major role in the successful diversification of metazoans, including our own human lineage. However, unlike other rapidly evolving functional systems such as sperm-egg interactions and immune defense, the genetic basis of rapid behavioral change remains elusive. Here we propose that the rapid divergence and widespread novelty of innate and adaptive behavior is primarily a function of its genomic architecture. Specifically, we hypothesize that the broad diversity of behavioral phenotypes present at micro- and macroevolutionary scales is promoted by a disproportionately large mutational target of neurogenic genes. We present evidence that these large neuro-behavioral targets are significant and ubiquitous in animal genomes and suggest that behavior's novelty and rapid emergence are driven by a number of factors including more selection on a larger pool of variants, a greater role of phenotypic plasticity, and/or unique molecular features present in large genes. We briefly discuss the origins of these large neurogenic genes, as they relate to the remarkable diversity of metazoan behaviors, and highlight key consequences on both behavioral traits and neurogenic disease across, respectively, evolutionary and ontogenetic time scales. Current approaches to studying the genetic mechanisms underlying rapid phenotypic change primarily focus on identifying signatures of Darwinian selection in protein-coding regions. In contrast

  1. The identification of irreversible rituximab-resistant lymphoma caused by CD20 gene mutations

    Energy Technology Data Exchange (ETDEWEB)

    Mishima, Y [Department of Clinical Chemotherapy, Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, Tokyo (Japan); Olympas Bio-Imaging Lab, Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, Tokyo (Japan); Terui, Y [Department of Clinical Chemotherapy, Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, Tokyo (Japan); Takeuchi, K [Division of Pathology, Cancer Institute, Japanese Foundation for Cancer Research, Tokyo (Japan); Matsumoto-Mishima, Y; Matsusaka, S [Department of Clinical Chemotherapy, Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, Tokyo (Japan); Utsubo-Kuniyoshi, R [Department of Clinical Chemotherapy, Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, Tokyo (Japan); Olympas Bio-Imaging Lab, Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, Tokyo (Japan); Hatake, K [Department of Clinical Chemotherapy, Cancer Chemotherapy Center, Japanese Foundation for Cancer Research, Tokyo (Japan)

    2011-04-01

    C-terminal mutations of CD20 constitute part of the mechanisms that resist rituximab therapy. Most CD20 having a C-terminal mutation was not recognized by L26 antibody. As the exact epitope of L26 has not been determined, expression and localization of mutated CD20 have not been completely elucidated. In this study, we revealed that the binding site of L26 monoclonal antibody is located in the C-terminal cytoplasmic region of CD20 molecule, which was often lost in mutated CD20 molecules. This indicates that it is difficult to distinguish the mutation of CD20 from under expression of the CD20 protein. To detect comprehensive CD20 molecules including the resistant mutants, we developed a novel monoclonal antibody that recognizes the N-terminal cytoplasm region of CD20 molecule. We screened L26-negative cases with our antibody and found several mutations. A rituximab-binding analysis using the cryopreserved specimen that mutation was identified in CD20 molecules indicated that the C-terminal region of CD20 undertakes a critical role in presentation of the large loop in which the rituximab-binding site locates. Thus, combination of antibodies of two kinds of epitope permits the identification of C-terminal CD20 mutations associated with irreversible resistance to rituximab and may help the decision of the treatment strategy.

  2. ATP1A3 Mutation in Adult Rapid-Onset Ataxia.

    Directory of Open Access Journals (Sweden)

    Kathleen J Sweadner

    Full Text Available A 21-year old male presented with ataxia and dysarthria that had appeared over a period of months. Exome sequencing identified a de novo missense variant in ATP1A3, the gene encoding the α3 subunit of Na,K-ATPase. Several lines of evidence suggest that the variant is causative. ATP1A3 mutations can cause rapid-onset dystonia-parkinsonism (RDP with a similar age and speed of onset, as well as severe diseases of infancy. The patient's ATP1A3 p.Gly316Ser mutation was validated in the laboratory by the impaired ability of the expressed protein to support the growth of cultured cells. In a crystal structure of Na,K-ATPase, the mutated amino acid was directly apposed to a different amino acid mutated in RDP. Clinical evaluation showed that the patient had many characteristics of RDP, however he had minimal fixed dystonia, a defining symptom of RDP. Successive magnetic resonance imaging (MRI revealed progressive cerebellar atrophy, explaining the ataxia. The absence of dystonia in the presence of other RDP symptoms corroborates other evidence that the cerebellum contributes importantly to dystonia pathophysiology. We discuss the possibility that a second de novo variant, in ubiquilin 4 (UBQLN4, a ubiquitin pathway component, contributed to the cerebellar neurodegenerative phenotype and differentiated the disease from other manifestations of ATP1A3 mutations. We also show that a homozygous variant in GPRIN1 (G protein-regulated inducer of neurite outgrowth 1 deletes a motif with multiple copies and is unlikely to be causative.

  3. Identification of coding and non-coding mutational hotspots in cancer genomes.

    Science.gov (United States)

    Piraino, Scott W; Furney, Simon J

    2017-01-05

    The identification of mutations that play a causal role in tumour development, so called "driver" mutations, is of critical importance for understanding how cancers form and how they might be treated. Several large cancer sequencing projects have identified genes that are recurrently mutated in cancer patients, suggesting a role in tumourigenesis. While the landscape of coding drivers has been extensively studied and many of the most prominent driver genes are well characterised, comparatively less is known about the role of mutations in the non-coding regions of the genome in cancer development. The continuing fall in genome sequencing costs has resulted in a concomitant increase in the number of cancer whole genome sequences being produced, facilitating systematic interrogation of both the coding and non-coding regions of cancer genomes. To examine the mutational landscapes of tumour genomes we have developed a novel method to identify mutational hotspots in tumour genomes using both mutational data and information on evolutionary conservation. We have applied our methodology to over 1300 whole cancer genomes and show that it identifies prominent coding and non-coding regions that are known or highly suspected to play a role in cancer. Importantly, we applied our method to the entire genome, rather than relying on predefined annotations (e.g. promoter regions) and we highlight recurrently mutated regions that may have resulted from increased exposure to mutational processes rather than selection, some of which have been identified previously as targets of selection. Finally, we implicate several pan-cancer and cancer-specific candidate non-coding regions, which could be involved in tumourigenesis. We have developed a framework to identify mutational hotspots in cancer genomes, which is applicable to the entire genome. This framework identifies known and novel coding and non-coding mutional hotspots and can be used to differentiate candidate driver regions from

  4. Cell cultures in uterine leiomyomas: rapid disappearance of cells carrying MED12 mutations.

    Science.gov (United States)

    Nadine Markowski, Dominique; Tadayyon, Mahboobeh; Bartnitzke, Sabine; Belge, Gazanfer; Maria Helmke, Burkhard; Bullerdiek, Jörn

    2014-04-01

    Uterine leiomyomas (UL) are the most frequent symptomatic human tumors. Nevertheless, their molecular pathogenesis is not yet fully understood. To learn more about the biology of these common neoplasms and their response to treatment, cell cultures derived from UL are a frequently used model system, but until recently appropriate genetic markers confirming their origin from the tumor cell population were lacking for most UL, i.e., those not displaying karyotypic abnormalities. The identification of MED12 mutations in the majority of UL makes it possible to trace the tumor cell population during in vitro passaging in the absence of cytogenetic abnormalities. The present study is addressing the in vitro survival of cells carrying MED12 mutations and its association with karyotypic alterations. The results challenge numerous in vitro studies into the biology and behavior of leiomyomas. Cells of one genetic subtype of UL, i.e., those with rearrangements of the high mobility AT-hook 2 protein gene (HMGA2), seem to be able to proliferate in vitro for many passages whereas tumor cells from the much more frequent MED12-mutated lesions barely survive even the first passages. Apparently, for the most frequent type of human UL no good in vitro model seems to exist because cells do not survive culturing. On the other hand, this inability may point to an Achilles' heel of this type of UL. Copyright © 2014 Wiley Periodicals, Inc.

  5. Identification of a novel STAT3 mutation in a patient with hyper-IgE syndrome

    DEFF Research Database (Denmark)

    Mogensen, Trine H; Jakobsen, Marianne; Larsen, Carsten S

    2013-01-01

    Here we describe a patient with hyper-IgE syndrome presenting with recurrent staphylococcal abscesses, pneumonia, and chronic mucocutaneous candidiasis, and report the identification of a novel STAT3 mutation at amino acid position 621, which has not previously been described. In addition, we...

  6. Identification of a novel SHOX mutation in a Chinese family with ...

    Indian Academy of Sciences (India)

    [Mei L., Huang Y., Pan Q., Li H., Liang D. and Wu L. 2014 Identification of a novel SHOX mutation in a Chinese family with isolated. Madelung deformity. J. Genet. ... females than males (Lichtenstein et al. 1980; Grigelioniene et al. 2001). .... proteins play critical roles in controlling the embryonic development in vertebrates ...

  7. Rapid method for identification of transgenic fish zygosity

    Directory of Open Access Journals (Sweden)

    . Alimuddin

    2007-07-01

    Full Text Available Identification of zygosity in transgenik fish is normally achieved by PCR analysis with genomic DNA template extracted from the tissue of progenies which are derived by mating the transgenic fish and wild-type counterpart.  This method needs relatively large amounts of fish material and is time- and labor-intensive. New approaches addressing this problem could be of great help for fish biotechnologists.  In this experiment, we applied a quantitative real-time PCR (qr-PCR method to analyze zygosity in a stable line of transgenic zebrafish (Danio rerio carrying masu salmon, Oncorhynchus masou D6-desaturase-like gene. The qr-PCR was performed using iQ SYBR Green Supermix in the iCycler iQ Real-time PCR Detection System (Bio-Rad Laboratories, USA.  Data were analyzed using the comparative cycle threshold method.  The results demonstrated a clear-cut identification of all transgenic fish (n=20 classified as a homozygous or heterozygous.  Mating of those fish with wild-type had revealed transgene transmission to the offspring following expected Mendelian laws. Thus, we found that the qTR-PCR to be effective for a rapid and precise determination of zygosity in transgenic fish. This technique could be useful in the establishment of breeding programs for mass transgenic fish production and in experiments in which zygosity effect could have a functional impact. Keywords: quantitative real-time PCR; zygosity; transgenic fish; mass production   ABSTRAK Identifikasi sigositas ikan transgenik biasanya dilakukan menggunakan analisa PCR dengan cetakan DNA genomik yang diekstraksi dari jaringan ikan hasil persilangan antara ikan transgenik dan ikan normal.   Metode ini memerlukan ikan dalam jumlah yang banyak, dan juga waktu serta tenaga.  Pendekatan baru untuk mengatasi masalah tersebut akan memberikan manfaat besar kepada peneliti bioteknologi perikanan.  Pada penelitian ini, kami menggunakan metode PCR real-time kuantitatif (krt-PCR untuk

  8. Rapid screening for nuclear genes mutations in isolated respiratory chain complex I defects.

    Science.gov (United States)

    Pagniez-Mammeri, Hélène; Lombes, Anne; Brivet, Michèle; Ogier-de Baulny, Hélène; Landrieu, Pierre; Legrand, Alain; Slama, Abdelhamid

    2009-04-01

    Complex I or reduced nicotinamide adenine dinucleotide (NADH): ubiquinone oxydoreductase deficiency is the most common cause of respiratory chain defects. Molecular bases of complex I deficiencies are rarely identified because of the dual genetic origin of this multi-enzymatic complex (nuclear DNA and mitochondrial DNA) and the lack of phenotype-genotype correlation. We used a rapid method to screen patients with isolated complex I deficiencies for nuclear genes mutations by Surveyor nuclease digestion of cDNAs. Eight complex I nuclear genes, among the most frequently mutated (NDUFS1, NDUFS2, NDUFS3, NDUFS4, NDUFS7, NDUFS8, NDUFV1 and NDUFV2), were studied in 22 cDNA fragments spanning their coding sequences in 8 patients with a biochemically proved complex I deficiency. Single nucleotide polymorphisms and missense mutations were detected in 18.7% of the cDNA fragments by Surveyor nuclease treatment. Molecular defects were detected in 3 patients. Surveyor nuclease screening is a reliable method for genotyping nuclear complex I deficiencies, easy to interpret, and limits the number of sequence reactions. Its use will enhance the possibility of prenatal diagnosis and help us for a better understanding of complex I molecular defects.

  9. Mutation Spectrum of the ABCA4 Gene in a Greek Cohort with Stargardt Disease: Identification of Novel Mutations and Evidence of Three Prevalent Mutated Alleles

    Directory of Open Access Journals (Sweden)

    Kamakari Smaragda

    2018-01-01

    Full Text Available Aim. To evaluate the frequency and pattern of disease-associated mutations of ABCA4 gene among Greek patients with presumed Stargardt disease (STGD1. Materials and Methods. A total of 59 patients were analyzed for ABCA4 mutations using the ABCR400 microarray and PCR-based sequencing of all coding exons and flanking intronic regions. MLPA analysis as well as sequencing of two regions in introns 30 and 36 reported earlier to harbor deep intronic disease-associated variants was used in 4 selected cases. Results. An overall detection rate of at least one mutant allele was achieved in 52 of the 59 patients (88.1%. Direct sequencing improved significantly the complete characterization rate, that is, identification of two mutations compared to the microarray analysis (93.1% versus 50%. In total, 40 distinct potentially disease-causing variants of the ABCA4 gene were detected, including six previously unreported potentially pathogenic variants. Among the disease-causing variants, in this cohort, the most frequent was c.5714+5G>A representing 16.1%, while p.Gly1961Glu and p.Leu541Pro represented 15.2% and 8.5%, respectively. Conclusions. By using a combination of methods, we completely molecularly diagnosed 48 of the 59 patients studied. In addition, we identified six previously unreported, potentially pathogenic ABCA4 mutations.

  10. A Comparison Between Denaturing Gradient Gel Electrophoresis and Denaturing High Performance Liquid Chromatography in Detecting Mutations in Genes Associated with Hereditary Non-Polyposis Colorectal Cancer (HNPCC and the Identification of 9 New Mutations Previously Unidentified by DGGE

    Directory of Open Access Journals (Sweden)

    Meldrum Cliff J

    2003-12-01

    Full Text Available Abstract Denaturing high performance liquid chromatography is a relatively new method by which heteroduplex structures formed during the PCR amplification of heterozygote samples can be rapidly identified. The use of this technology for mutation detection in hereditary non-polyposis colorectal cancer (HNPCC has the potential to appreciably shorten the time it takes to analyze genes associated with this disorder. Prior to acceptance of this method for screening genes associated with HNPCC, assessment of the reliability of this method should be performed. In this report we have compared mutation and polymorphism detection by denaturing gradient gel electrophoresis (DGGE with denaturing high performance liquid chromatography (DHPLC in a set of 130 families. All mutations/polymorphisms representing base substitutions, deletions, insertions and a 23 base pair inversion were detected by DHPLC whereas DGGE failed to identify four single base substitutions and a single base pair deletion. In addition, we show that DHPLC has been used for the identification of 5 different mutations in exon 7 of hMSH2 that could not be detected by DGGE. From this study we conclude that DHPLC is a more effective and rapid alternative to the detection of mutations in hMSH2 and hMLH1 with the same or better accuracy than DGGE. Furthermore, this technique offers opportunities for automation, which have not been realised for the majority of other methods of gene analysis.

  11. Identification of Novel Mutations in FAH Gene and Prenatal Diagnosis of Tyrosinemia in Indian Family

    Directory of Open Access Journals (Sweden)

    Jayesh J. Sheth

    2012-01-01

    Full Text Available Carrier of tyrosinemia type I was diagnosed by sequencing FAH (fumarylacetoacetate hydrolase gene. It leads to the identification of heterozygous status for both c.648C>G (p.Ile216Met and c.1159G>A (p.Gly387Arg mutations in exons 8 and 13, respectively, in the parents. The experimental program PolyPhen, SIFT, and MT predicts former missense point mutation as “benign” that creates a potential donor splice site and later one as “probably damaging” which disrupts secondary structure of protein.

  12. Rapid Electrochemical Detection and Identification of Microbiological and Chemical Contaminants for Manned Spaceflight Project

    Data.gov (United States)

    National Aeronautics and Space Administration — A great deal of effort has gone into the development of point-of-use methods to meet the challenge of rapid bacterial identification for both environmental...

  13. Identification of seven novel mutations including the first two genomic rearrangements in SLC26A3 mutated in congenital chloride diarrhea.

    Science.gov (United States)

    Höglund, P; Sormaala, M; Haila, S; Socha, J; Rajaram, U; Scheurlen, W; Sinaasappel, M; de Jonge, H; Holmberg, C; Yoshikawa, H; Kere, J

    2001-09-01

    Congenital chloride diarrhea (CLD) is an autosomal recessive disorder characterized by defective intestinal electrolyte absorption, resulting in voluminous osmotic diarrhea with high chloride content. A variety of mutations in the solute carrier family 26, member 3 gene (SLC26A3, previously known as CLD or DRA) are responsible for the disease. Since the identification of the SLC26A3 gene and the determination of its genomic structure, altogether three founder and 17 private mutations have been characterized within miscellaneous ethnic groups. We screened for mutations in seven unrelated families with CLD. The diagnoses were confirmed by fecal chloride measurements. The combined PCR-SSCP and sequencing analyses revealed altogether seven novel mutations including two missense mutations (S206P, D468V), two splicing defects (IVS12-1G>C, IVS13-2delA), one nonsense mutation (Q436X), one insertion/deletion mutation (2104-2105delGGins29-bp), and an intragenic deletion of SLC26A3 exons 7 and 8. Two previously identified mutations were also found. This is the first report of rearrangement mutations in SLC26A3. Molecular features predisposing SLC26A3 for the two rearrangements may include repetitive elements and palindromic-like sequences. The increasingly wide diversity of SLC26A3 mutations suggests that mutations in the SLC26A3 gene may not be rare events. Copyright 2001 Wiley-Liss, Inc.

  14. Rapid identification and detection of pathogenic Fungi by padlock probes

    NARCIS (Netherlands)

    Tsui, C.K.M.; Wang, B.; Schoen, C.D.; Hamelin, R.C.

    2013-01-01

    Fungi are important pathogens of human diseases, as well as to agricultural crop and trees. Molecular diagnostics can detect diseases early, and improve identification accuracy and follow-up disease management. The use of padlock probe is effective to facilitate these detections and pathogen

  15. Rapid identification of the medicinal plant Taraxacum formosanum ...

    African Journals Online (AJOL)

    Original identification of medicinal plants is essential for quality control. In this study, the internal transcribed spacer 2 (ITS2) nuclear ribosomal DNA served as a DNA barcode and was amplified by allele-specific PCR. This approach was exploited to differentiate Taraxacum formosanum from five related adulterants. Using a ...

  16. On-site identification of meat species in processed foods by a rapid real-time polymerase chain reaction system.

    Science.gov (United States)

    Furutani, Shunsuke; Hagihara, Yoshihisa; Nagai, Hidenori

    2017-09-01

    Correct labeling of foods is critical for consumers who wish to avoid a specific meat species for religious or cultural reasons. Therefore, gene-based point-of-care food analysis by real-time Polymerase Chain Reaction (PCR) is expected to contribute to the quality control in the food industry. In this study, we perform rapid identification of meat species by our portable rapid real-time PCR system, following a very simple DNA extraction method. Applying these techniques, we correctly identified beef, pork, chicken, rabbit, horse, and mutton in processed foods in 20min. Our system was sensitive enough to detect the interfusion of about 0.1% chicken egg-derived DNA in a processed food sample. Our rapid real-time PCR system is expected to contribute to the quality control in food industries because it can be applied for the identification of meat species, and future applications can expand its functionality to the detection of genetically modified organisms or mutations. Copyright © 2017 Elsevier Ltd. All rights reserved.

  17. Identification of unique repeated patterns, location of mutation in DNA finger printing using artificial intelligence technique.

    Science.gov (United States)

    Mukunthan, B; Nagaveni, N

    2014-01-01

    In genetic engineering, conventional techniques and algorithms employed by forensic scientists to assist in identification of individuals on the basis of their respective DNA profiles involves more complex computational steps and mathematical formulae, also the identification of location of mutation in a genomic sequence in laboratories is still an exigent task. This novel approach provides ability to solve the problems that do not have an algorithmic solution and the available solutions are also too complex to be found. The perfect blend made of bioinformatics and neural networks technique results in efficient DNA pattern analysis algorithm with utmost prediction accuracy.

  18. Rapid screening of rpoB and katG mutations in Mycobacterium tuberculosis isolates by high-resolution melting curve analysis

    Directory of Open Access Journals (Sweden)

    M Haeili

    2014-01-01

    Full Text Available Background: Early detection of multidrug-resistant tuberculosis (MDR-TB is essential to prevent its transmission in the community and initiate effective anti-TB treatment regimen. Materials and Methods: High-resolution melting curve (HRM analysis was evaluated for rapid detection of resistance conferring mutations in rpoB and katG genes. We screened 95 Mycobacterium tuberculosis clinical isolates including 20 rifampin resistant (RIF-R, 21 isoniazid resistant (INH-R and 54 fully susceptible (S isolates determined by proportion method of drug susceptibility testing. Nineteen M. tuberculosis isolates with known drug susceptibility genotypes were used as references for the assay validation. The nucleotide sequences of the target regions rpoB and katG genes were determined to investigate the frequency and type of mutations and to confirm HRM results. Results: HRM analysis of a 129-bp fragment of rpoB allowed correct identification of 19 of the 20 phenotypically RIF-R and all RIF-S isolates. All INH-S isolates generated wild-type HRM curves and 18 out of 21 INH-R isolates harboured any mutation in 109-bp fragment of katG exhibited mutant type HRM curves. However, 1 RIF-R and 3 INH-R isolates were falsely identified as susceptible which were confirmed for having no mutation in their target regions by sequencing. The main mutations involved in RIF and INH resistance were found at codons rpoB531 (60% of RIF-R isolates and katG315 (85.7% of INH-R isolates, respectively. Conclusion: HRM was found to be a reliable, rapid and low cost method to characterise drug susceptibility of clinical TB isolates in resource-limited settings.

  19. Rapidly progressive renal disease as part of Wolfram syndrome in a large inbred Turkish family due to a novel WFS1 mutation (p.Leu511Pro).

    Science.gov (United States)

    Yuca, Sevil Ari; Rendtorff, Nanna Dahl; Boulahbel, Houda; Lodahl, Marianne; Tranebjærg, Lisbeth; Cesur, Yasar; Dogan, Murat; Yilmaz, Cahide; Akgun, Cihangir; Acikgoz, Mehmet

    2012-01-01

    Wolfram syndrome, also named "DIDMOAD" (diabetes insipidus, diabetes mellitus, optic atrophy, and deafness), is an inherited association of juvenile-onset diabetes mellitus and optic atrophy as key diagnostic criteria. Renal tract abnormalities and neurodegenerative disorder may occur in the third and fourth decade. The wolframin gene, WFS1, associated with this syndrome, is located on chromosome 4p16.1. Many mutations have been described since the identification of WFS1 as the cause of Wolfram syndrome. We identified a new homozygous WFS1 mutation (c.1532T>C; p.Leu511Pro) causing Wolfram syndrome in a large inbred Turkish family. The patients showed early onset of IDDM, diabetes insipidus, optic atrophy, sensorineural hearing impairment and very rapid progression to renal failure before age 12 in three females. Ectopic expression of the wolframin mutant in HEK cells results in greatly reduced levels of protein expression compared to wild-type wolframin, strongly supporting that this mutation is disease-causing. The mutation showed perfect segregation with disease in the family, characterized by early and severe clinical manifestations. Copyright © 2011 Elsevier Masson SAS. All rights reserved.

  20. Rapid targeted somatic mutation analysis of solid tumors in routine clinical diagnostics.

    Science.gov (United States)

    Magliacane, Gilda; Grassini, Greta; Bartocci, Paola; Francaviglia, Ilaria; Dal Cin, Elena; Barbieri, Gianluca; Arrigoni, Gianluigi; Pecciarini, Lorenza; Doglioni, Claudio; Cangi, Maria Giulia

    2015-10-13

    Tumor genotyping is an essential step in routine clinical practice and pathology laboratories face a major challenge in being able to provide rapid, sensitive and updated molecular tests. We developed a novel mass spectrometry multiplexed genotyping platform named PentaPanel to concurrently assess single nucleotide polymorphisms in 56 hotspots of the 5 most clinically relevant cancer genes, KRAS, NRAS, BRAF, EGFR and PIK3CA for a total of 221 detectable mutations. To both evaluate and validate the PentaPanel performance, we investigated 1025 tumor specimens of 6 different cancer types (carcinomas of colon, lung, breast, pancreas, and biliary tract, and melanomas), systematically addressing sensitivity, specificity, and reproducibility of our platform. Sanger sequencing was also performed for all the study samples. Our data showed that PentaPanel is a high throughput and robust tool, allowing genotyping for targeted therapy selection of 10 patients in the same run, with a practical turnaround time of 2 working days. Importantly, it was successfully used to interrogate different DNAs isolated from routinely processed specimens (formalin-fixed paraffin embedded, frozen, and cytological samples), covering all the requirements of clinical tests. In conclusion, the PentaPanel platform can provide an immediate, accurate and cost effective multiplex approach for clinically relevant gene mutation analysis in many solid tumors and its utility across many diseases can be particularly relevant in multiple clinical trials, including the new basket trial approach, aiming to identify appropriate targeted drug combination strategies.

  1. Identification of mutated driver pathways in cancer using a multi-objective optimization model.

    Science.gov (United States)

    Zheng, Chun-Hou; Yang, Wu; Chong, Yan-Wen; Xia, Jun-Feng

    2016-05-01

    New-generation high-throughput technologies, including next-generation sequencing technology, have been extensively applied to solve biological problems. As a result, large cancer genomics projects such as the Cancer Genome Atlas (TCGA) and the International Cancer Genome Consortium are producing large amount of rich and diverse data in multiple cancer types. The identification of mutated driver genes and driver pathways from these data is a significant challenge. Genome aberrations in cancer cells can be divided into two types: random 'passenger mutation' and functional 'driver mutation'. In this paper, we introduced a Multi-objective Optimization model based on a Genetic Algorithm (MOGA) to solve the maximum weight submatrix problem, which can be employed to identify driver genes and driver pathways promoting cancer proliferation. The maximum weight submatrix problem defined to find mutated driver pathways is based on two specific properties, i.e., high coverage and high exclusivity. The multi-objective optimization model can adjust the trade-off between high coverage and high exclusivity. We proposed an integrative model by combining gene expression data and mutation data to improve the performance of the MOGA algorithm in a biological context. Copyright © 2016 Elsevier Ltd. All rights reserved.

  2. New Technologies for Rapid Bacterial Identification and Antibiotic Resistance Profiling.

    Science.gov (United States)

    Kelley, Shana O

    2017-04-01

    Conventional approaches to bacterial identification and drug susceptibility testing typically rely on culture-based approaches that take 2 to 7 days to return results. The long turnaround times contribute to the spread of infectious disease, negative patient outcomes, and the misuse of antibiotics that can contribute to antibiotic resistance. To provide new solutions enabling faster bacterial analysis, a variety of approaches are under development that leverage single-cell analysis, microfluidic concentration and detection strategies, and ultrasensitive readout mechanisms. This review discusses recent advances in this area and the potential of new technologies to enable more effective management of infectious disease.

  3. Rapid identification of drugs in the overdosed patient.

    Science.gov (United States)

    Hackett, L P; Dusci, L J

    1977-01-01

    A rapid analytical procedure is described for a variety of drugs that could be present in the overdosed patient. The technique used gives quantitative results for most of the drugs analyzed in serum using gas chromatography and incorporates thin-layer chromatography and spot tests for drug confirmation. The procedure is novel for it relies on the initial extraction of acidics, basics, and neutrals from serum acidified with hydroxhloric acid.

  4. Mutational profiles of breast cancer metastases from a rapid autopsy series reveal multiple evolutionary trajectories.

    Science.gov (United States)

    Avigdor, Bracha Erlanger; Cimino-Mathews, Ashley; DeMarzo, Angelo M; Hicks, Jessica L; Shin, James; Sukumar, Saraswati; Fetting, John; Argani, Pedram; Park, Ben H; Wheelan, Sarah J

    2017-12-21

    Heterogeneity within and among tumors in a metastatic cancer patient is a well-established phenomenon that may confound treatment and accurate prognosis. Here, we used whole-exome sequencing to survey metastatic breast cancer tumors from 5 patients in a rapid autopsy program to construct the origin and genetic development of metastases. Metastases were obtained from 5 breast cancer patients using a rapid autopsy protocol and subjected to whole-exome sequencing. Metastases were evaluated for sharing of somatic mutations, correlation of copy number variation and loss of heterozygosity, and genetic similarity scores. Pathological features of the patients' disease were assessed by immunohistochemical analyses. Our data support a monoclonal origin of metastasis in 3 cases, but in 2 cases, metastases arose from at least 2 distinct subclones in the primary tumor. In the latter 2 cases, the primary tumor presented with mixed histologic and pathologic features, suggesting early divergent evolution within the primary tumor with maintenance of metastatic capability in multiple lineages. We used genetic and histopathological evidence to demonstrate that metastases can be derived from a single or multiple independent clones within a primary tumor. This underscores the complexity of breast cancer clonal evolution and has implications for how best to determine and implement therapies for early- and late-stage disease.

  5. Physical Mapping Technologies for the Identification and Characterization of Mutated Genes to Crop Quality

    International Nuclear Information System (INIS)

    2011-09-01

    The improvement of quality traits in food and industrial crops is an important breeding objective for both developed and developing countries in order to add value to the crop and thereby increasing farmers' income. It has been well established that the application of mutagens can be a very important approach for manipulating many crop characteristics including quality. While mutation induction using nuclear techniques such as gamma irradiation is a power tool in generating new genotypes with favourable alleles for improving crop quality in plant breeding, a more thorough understanding of gene expression, gene interactions, and physical location will improve ability to manipulate and control genes, and directly lead to crop improvement. Physical mapping technologies, molecular markers and molecular cytogenetic techniques are tools available with the potential to enhance the ability to tag genes and gene complexes to facilitate the selection of desirable genotypes in breeding programmes, including those based on mutation breeding. This Coordinated Research Project (CRP) on 'Physical Mapping Technologies for the Identification and Characterization of Mutated Genes Contributing to Crop Quality' was conducted under the overall IAEA project objective of 'Identification, Characterization and Transfer of Mutated Genes'. The specific objectives of the CRP were to assist Member States in accelerating crop breeding programmes through the application of physical mapping and complementary genomic approaches, and the characterization and utilization of induced mutants for improvement of crop quality. The IAEA-TECDOC describes the success obtained in the application of molecular cytology, molecular markers, physical mapping and mutation technologies since the inception of the CRP in 2003. The CRP also resulted in two book chapters, 35 peer reviewed papers, 25 conference proceedings, one PhD thesis, and 22 published abstracts. In addition, thirteen sequences were submitted to the

  6. A rapid, one step molecular identification of Trichoderma citrinoviride and Trichoderma reesei.

    Science.gov (United States)

    Saroj, Dina B; Dengeti, Shrinivas N; Aher, Supriya; Gupta, Anil K

    2015-06-01

    Trichoderma species are widely used as production hosts for industrial enzymes. Identification of Trichoderma species requires a complex molecular biology based identification involving amplification and sequencing of multiple genes. Industrial laboratories are required to run identification tests repeatedly in cell banking procedures and also to prove absence of production host in the product. Such demands can be fulfilled by a brief method which enables confirmation of strain identity. This communication describes one step identification method for two common Trichoderma species; T. citrinoviride and T. reesei, based on identification of polymorphic region in the nucleotide sequence of translation elongation factor 1 alpha. A unique forward primer and common reverse primer resulted in 153 and 139 bp amplicon for T. citrinoviride and T. reesei, respectively. Simplification was further introduced by using mycelium as template for PCR amplification. Method described in this communication allows rapid, one step identification of two Trichoderma species.

  7. [Rapid identification of hogwash oil by using synchronous fluorescence spectroscopy].

    Science.gov (United States)

    Sun, Yan-Hui; An, Hai-Yang; Jia, Xiao-Li; Wang, Juan

    2012-10-01

    To identify hogwash oil quickly, the characteristic delta lambda of hogwash oil was analyzed by three dimensional fluorescence spectroscopy with parallel factor analysis, and the model was built up by using synchronous fluorescence spectroscopy with support vector machines (SVM). The results showed that the characteristic delta lambda of hogwash oil was 60 nm. Collecting original spectrum of different samples under the condition of characteristic delta lambda 60 nm, the best model was established while 5 principal components were selected from original spectrum and the radial basis function (RBF) was used as the kernel function, and the optimal penalty factor C and kernel function g were 512 and 0.5 respectively obtained by the grid searching and 6-fold cross validation. The discrimination rate of the model was 100% for both training sets and prediction sets. Thus, it is quick and accurate to apply synchronous fluorescence spectroscopy to identification of hogwash oil.

  8. High resolution melting curve analysis, a rapid and affordable method for mutation analysis in childhood acute myeloid leukemia

    Directory of Open Access Journals (Sweden)

    Yin eLiu

    2014-09-01

    Full Text Available Background: Molecular genetic alterations with prognostic significance have been described in childhood acute myeloid leukemia (AML. The aim of this study was to establish cost-effective techniques to detect mutations of FMS-like tyrosine kinase 3 (FLT3, Nucleophosmin 1 (NPM1, and a partial tandem duplication within the mixed lineage leukemia (MLL-PTD genes in childhood AML. Procedure: Ninety-nine children with newly diagnosed AML were included in this study. We developed a fluoresent dye SYTO-82 based high resolution melting curve (HRM anaylsis to detect FLT3 internal tandem duplication (FLT3-ITD, FLT3 tyrosine kinase domain (FLT3-TKD and NPM1 mutations. MLL-PTD was screened by real-time quantitative PCR. Results: The HRM methodology correlated well with gold standard Sanger sequencing with less cost. Among the 99 patients studied, the FLT3-ITD mutation was associated with significantly worse event free survival (EFS. Patients with the NPM1 mutation had significantly better EFS and overall survival. However, HRM was not sensitive enough for minimal residual disease monitoring. Conclusions: HRM was a rapid and efficient method for screening of FLT3 and NPM1 gene mutations. It was both affordable and accurate, especially in resource underprivileged regions. Our results indicated that HRM could be a useful clinical tool for rapid and cost effective screening of the FLT3 and NPM1 mutations in AML patients.

  9. Small acid soluble proteins for rapid spore identification.

    Energy Technology Data Exchange (ETDEWEB)

    Branda, Steven S.; Lane, Todd W.; VanderNoot, Victoria A.; Jokerst, Amanda S.

    2006-12-01

    This one year LDRD addressed the problem of rapid characterization of bacterial spores such as those from the genus Bacillus, the group that contains pathogenic spores such as B. anthracis. In this effort we addressed the feasibility of using a proteomics based approach to spore characterization using a subset of conserved spore proteins known as the small acid soluble proteins or SASPs. We proposed developing techniques that built on our previous expertise in microseparations to rapidly characterize or identify spores. An alternative SASP extraction method was developed that was amenable to both the subsequent fluorescent labeling required for laser-induced fluorescence detection and the low ionic strength requirements for isoelectric focusing. For the microseparations, both capillary isoelectric focusing and chip gel electrophoresis were employed. A variety of methods were evaluated to improve the molecular weight resolution for the SASPs, which are in a molecular weight range that is not well resolved by the current methods. Isoelectric focusing was optimized and employed to resolve the SASPs using UV absorbance detection. Proteomic signatures of native wild type Bacillus spores and clones genetically engineered to produce altered SASP patterns were assessed by slab gel electrophoresis, capillary isoelectric focusing with absorbance detection as well as microchip based gel electrophoresis employing sensitive laser-induced fluorescence detection.

  10. A rapid, sensitive, reproducible and cost-effective method for mutation profiling of colon cancer and metastatic lymph nodes

    International Nuclear Information System (INIS)

    Fumagalli, Debora; Gavin, Patrick G; Taniyama, Yusuke; Kim, Seung-Il; Choi, Hyun-Joo; Paik, Soonmyung; Pogue-Geile, Katherine L

    2010-01-01

    Carta panel may provide a mechanism for rapid screening of mutations in clinically relevant genes like KRAS, PIK3CA, and BRAF. ClinicalTrials.gov: NSABP C-07: NCT00004931

  11. Rapidly Learned Identification of Epileptic Seizures from Sonified EEG

    Directory of Open Access Journals (Sweden)

    Psyche eLoui

    2014-10-01

    Full Text Available Sonification refers to a process by which data are converted into sound, providing an auditory alternative to visual display. Currently, the prevalent method for diagnosing seizures in epilepsy is by visually reading a patient’s electroencephalogram (EEG. However, sonification of the EEG data provides certain advantages due to the nature of human auditory perception. We hypothesized that human listeners will be able to identify seizures from EEGs using the auditory modality alone, and that accuracy of seizure identification will increase after a short training session. Here we describe an algorithm we have used to sonify EEGs of both seizure and non-seizure activity, followed by a training study in which subjects listened to short clips of sonified EEGs and determine whether each clip was of seizure or normal activity, both before and after a short training session. Results show that before training subjects performed at chance level in differentiating seizures vs. non-seizures, but there was a significant improvement of accuracy after the training session. After training, subjects successfully distinguished seizures from non-seizures using the auditory modality alone. Further analyses using signal detection theory demonstrated improvement in sensitivity and reduction in response bias as a result of training. This study demonstrates the potential of sonified EEGs to be used for the detection of seizures. Future studies will attempt to increase accuracy using novel training and sonification modifications, with the goals of managing, predicting, and ultimately controlling seizures using sonification as a possible biofeedback-based intervention for epilepsy.

  12. Rapid detection of single nucleotide mutation in p53 gene based on ...

    Indian Academy of Sciences (India)

    mutation.27 Nevertheless, more than 50% of all human tumors contain p53 mutation; ... gene mutation detection in various fields of biology and medicine persuaded us to find ..... Yola M L, Eren T and Atar N 2014 Electrochim. Acta. 125 38. 26.

  13. Rapid Identification of Aldose Reductase Inhibitory Compounds from Perilla frutescens

    Directory of Open Access Journals (Sweden)

    Ji Hun Paek

    2013-01-01

    Full Text Available The ethyl acetate (EtOAc soluble fraction of methanol extracts of Perilla frutescens (P. frutescens inhibits aldose reductase (AR, the key enzyme in the polyol pathway. Our investigation of inhibitory compounds from the EtOAc soluble fraction of P. frutescens was followed by identification of the inhibitory compounds by a combination of HPLC microfractionation and a 96-well enzyme assay. This allowed the biological activities to be efficiently matched with selected HPLC peaks. Structural analyses of the active compounds were performed by LC-MSn. The main AR inhibiting compounds were tentatively identified as chlorogenic acid and rosmarinic acid by LC-MSn. A two-step high speed counter current chromatography (HSCCC isolation method was developed with a solvent system of n-hexane-ethyl acetate-methanol-water at 1.5 : 5 : 1 : 5, v/v and 3 : 7 : 5 : 5, v/v. The chemical structures of the isolated compounds were determined by 1H- and 13C-nuclear magnetic resonance spectrometry (NMR. The main compounds inhibiting AR in the EtOAc fraction of methanol extracts of P. frutescens were identified as chlorogenic acid (2 (IC50 = 3.16 μM, rosmarinic acid (4 (IC50 = 2.77 μM, luteolin (5 (IC50 = 6.34 μM, and methyl rosmarinic acid (6 (IC50 = 4.03 μM.

  14. Identification of a Likely Radio Counterpart to the Rapid Burster

    Science.gov (United States)

    Moore, Christopher B.; Rutledge, Robert E.; Fox, Derek W.; Guerriero, Robert A.; Lewin, Walter H. G.; Fender, Robert; van Paradijs, Jan

    2000-04-01

    We have identified a likely radio counterpart to the low-mass X-ray binary MXB 1730-335 (the Rapid Burster). The counterpart has shown 8.4 GHz radio on/off behavior correlated with the X-ray on/off behavior as observed by the RXTE/ASM during six VLA observations. The probability of an unrelated, randomly varying background source duplicating this behavior is 1%-3% depending on the correlation timescale. The location of the radio source is R.A. 17h33m24.61s, decl. -33 deg23'19.8" (J2000), +/-0.1". We do not detect 8.4 GHz radio emission coincident with type II (accretion-driven) X-ray bursts. The ratio of radio to X-ray emission during such bursts is constrained to be below the ratio observed during X-ray-persistent emission at the 2.9 σ level. Synchrotron bubble models of the radio emission can provide a reasonable fit to the full data set, collected over several outbursts, assuming that the radio evolution is the same from outburst to outburst but given the physical constraints the emission is more likely to be due to ~1 hr radio flares such as have been observed from the X-ray binary GRS 1915+105.

  15. Growth medium for the rapid isolation and identification of anthrax

    Science.gov (United States)

    Kiel, Johnathan L.; Parker, Jill E.; Grubbs, Teri R.; Alls, John L.

    2000-07-01

    Anthrax has been recognized as a highly likely biological warfare or terrorist agent. The purpose of this work was to design a culture technique to rapidly isolate and identify `live' anthrax. In liquid or solid media form, 3AT medium (3-amino-L-tyrosine, the main ingredient) accelerated germination and growth of anthrax spores in 5 to 6 hours to a point expected at 18 to 24 hours with ordinary medium. During accelerated growth, standard definitive diagnostic tests such as sensitivity to lysis by penicillin or bacteriophage can be run. During this time, the bacteria synthesized a fluorescent and thermochemiluminescent polymer. Bacteria captured by specific antibody are, therefore, already labeled. Because living bacteria are required to generate the polymer, the test converts immunoassays for anthrax into viability assays. Furthermore, the polymer formation leads to the death of the vegetative form and non-viability of the spores produced in the medium. By altering the formulation of the medium, other microbes and even animal and human cells can be grown in it and labeled (including viruses grown in the animal or human cells).

  16. A rapid PCR-based approach for molecular identification of filamentous fungi.

    Science.gov (United States)

    Chen, Yuanyuan; Prior, Bernard A; Shi, Guiyang; Wang, Zhengxiang

    2011-08-01

    In this study, a novel rapid and efficient DNA extraction method based on alkaline lysis, which can deal with a large number of filamentous fungal isolates in the same batch, was established. The filamentous fungal genomic DNA required only 20 min to prepare and can be directly used as a template for PCR amplification. The amplified internal transcribed spacer regions were easy to identify by analysis. The extracted DNA also can be used to amplify other protein-coding genes for fungal identification. This method can be used for rapid systematic identification of filamentous fungal isolates.

  17. Rapid identification of red-flesh loquat cultivars using EST-SSR markers based on manual cultivar identification diagram strategy.

    Science.gov (United States)

    Li, X Y; Xu, H X; Chen, J W

    2014-04-29

    Manual cultivar identification diagram is a new strategy for plant cultivar identification based on DNA markers, providing information to efficiently separate cultivars. We tested 25 pairs of apple EST-SSR primers for amplification of PCR products from loquat cultivars. These EST-SSR primers provided clear amplification products from the loquat cultivars, with a relatively high transferability rate of 84% to loquat; 11 pairs of primers amplified polymorphic products. After analysis of 24 red-fleshed loquat accessions, we found that only 7 pairs of primers could clearly separate all of them. A cultivar identification diagram of the 24 cultivars was constructed using polymorphic bands from the DNA fingerprints and EST-SSR primers. Any two of the 24 cultivars could be rapidly separated from each other, according to the polymorphic bands from the cultivars; the corresponding primers were marked in the correct position on the cultivar identification diagram. This red-flesh loquat cultivar identification diagram can separate the 24 red-flesh loquat cultivars, which is of benefit for loquat cultivar identification for germplasm management and breeding programs.

  18. Identification of a SIRT1 mutation in a family with type 1 diabetes

    DEFF Research Database (Denmark)

    Biason-Lauber, Anna; Böni-Schnetzler, Marianne; Hubbard, Basil P

    2013-01-01

    Type 1 diabetes is caused by autoimmune-mediated β cell destruction leading to insulin deficiency. The histone deacetylase SIRT1 plays an essential role in modulating several age-related diseases. Here we describe a family carrying a mutation in the SIRT1 gene, in which all five affected members...... developed an autoimmune disorder: four developed type 1 diabetes, and one developed ulcerative colitis. Initially, a 26-year-old man was diagnosed with the typical features of type 1 diabetes, including lean body mass, autoantibodies, T cell reactivity to β cell antigens, and a rapid dependence on insulin....... Direct and exome sequencing identified the presence of a T-to-C exchange in exon 1 of SIRT1, corresponding to a leucine-to-proline mutation at residue 107. Expression of SIRT1-L107P in insulin-producing cells resulted in overproduction of nitric oxide, cytokines, and chemokines. These observations...

  19. Time is of essence; rapid identification of veterinary pathogens using MALDI TOF

    DEFF Research Database (Denmark)

    Nonnemann, Bettina; Dalsgaard, Inger; Pedersen, Karl

    Rapid and accurate identification of microbial pathogens is a cornerstone for timely and correct treatment of diseases of livestock and fish. The utility of the MALDI-TOF technique in the diagnostic laboratory is directly related to the quality of mass spectra and quantity of different microbial...

  20. A rapid and direct real time PCR-based method for identification of Salmonella spp

    DEFF Research Database (Denmark)

    Rodriguez-Lazaro, D.; Hernández, Marta; Esteve, T.

    2003-01-01

    The aim of this work was the validation of a rapid, real-time PCR assay based on TaqMan((R)) technology for the unequivocal identification of Salmonella spp. to be used directly on an agar-grown colony. A real-time PCR system targeting at the Salmonella spp. invA gene was optimized and validated ...

  1. Identification of a nonsense mutation in CWC15 associated with decreased reproductive efficiency in Jersey cattle.

    Directory of Open Access Journals (Sweden)

    Tad S Sonstegard

    Full Text Available With the recent advent of genomic tools for cattle, several recessive conditions affecting fertility have been identified and selected against, such as deficiency of uridine monophosphate synthase, complex vertebral malformation, and brachyspina. The current report refines the location of a recessive haplotype affecting fertility in Jersey cattle using crossover haplotypes, discovers the causative mutation using whole genome sequencing, and examines the gene's role in embryo loss. In an attempt to identify unknown recessive lethal alleles in the current dairy population, a search using deep Mendelian sampling of 5,288 Jersey cattle was conducted for high-frequency haplotypes that have a deficit of homozygotes at the population level. This search led to the discovery of a putative recessive lethal in Jersey cattle on Bos taurus autosome 15. The haplotype, denoted JH1, was associated with reduced fertility, and further investigation identified one highly-influential Jersey bull as the putative source ancestor. By combining SNP analysis of whole-genome sequences aligned to the JH1 interval and subsequent SNP validation a nonsense mutation in CWC15 was identified as the likely causative mutation underlying the fertility phenotype. No homozygous recessive individuals were found in 749 genotyped animals, whereas all known carriers and carrier haplotypes possessed one copy of the mutant allele. This newly identified lethal has been responsible for a substantial number of spontaneous abortions in Jersey dairy cattle throughout the past half-century. With the mutation identified, selection against the deleterious allele in breeding schemes will aid in reducing the incidence of this defect in the population. These results also show that carrier status can be imputed with high accuracy. Whole-genome resequencing proved to be a powerful strategy to rapidly identify a previously mapped deleterious mutation in a known carrier of a recessive lethal allele.

  2. Comparison between MALDI-TOF MS and FilmArray Blood Culture Identification panel for rapid identification of yeast from positive blood culture.

    Science.gov (United States)

    Paolucci, M; Foschi, C; Tamburini, M V; Ambretti, S; Lazzarotto, T; Landini, M P

    2014-09-01

    In this study we evaluated MALDI-TOF MS and FilmArray methods for the rapid identification of yeast from positive blood cultures. FilmArray correctly identified 20/22 of yeast species, while MALDI-TOF MS identified 9/22. FilmArray is a reliable and rapid identification system for the direct identification of yeasts from positive blood cultures. Copyright © 2014 Elsevier B.V. All rights reserved.

  3. Rapid and accurate identification of Streptococcus equi subspecies by MALDI-TOF MS

    DEFF Research Database (Denmark)

    Kudirkiene, Egle; Welker, Martin; Knudsen, Nanna Reumert

    2015-01-01

    phenotypic and sequence similarity between three subspecies their discrimination remains difficult. In this study, we aimed to design and validate a novel, Superspectra based, MALDI-TOF MS approach for reliable, rapid and cost-effective identification of SEE and SEZ, the most frequent S. equi subspecies.......3±7.5%). This result may be attributed to the highly clonal population structure of SEE, as opposed to the diversity of SEZ seen in horses. Importantly strains with atypical colony appearance both within SEE and SEZ did not affect correct identification of the strains by MALDI-TOF MS. Atypical colony variants...... are often associated with a higher persistence or virulence of S. equi, thus their correct identification using the current method strengthens its potential use in routine clinical diagnostics. In conclusion, reliable identification of S. equi subspecies was achieved by combining a MALDI-TOF MS method...

  4. RAPID DETECTION OF -THALASSEMIA MUTATIONS IN THAILAND USING MULTIPLEX ARMS

    Directory of Open Access Journals (Sweden)

    D. Shimbhu

    2017-11-01

    Full Text Available The number of mutations underlining b-thalassemia generate a wide variety of different clinical phenotypes. An understanding of the genotype is important for medical personnel in order to provide proper counseling to patients and their families. Characterization of these mutations should aid the planning of a prenatal diagnosis program for bthalassemia. The heterogeneity of the mutations makes it difficult and time consuming to identify the mutation in some individuals. We developed a single-tube multiplex amplification refractory mutation system (multiplex ARMS to identify common ethnic- specific b-thalassemia mutations. Confirmation of multiplex ARMS results was carried out using direct sequencing. Three thousand three hundred twenty two people from Phitsanulok province were screened for the b-thalassemia trait by quantitation of HbA2 with microcolumn chromatography and the genotypes of mutations were characterized using multiplex ARMS and direct sequencing. We found that the deletion at codons 41/42 (-TCTT was the most frequent (48%, codon 17 (A®T (30%, -28 (A®G (6% and IVS-I-1(G®T (6% were the second and third in frequency respectively. A -87 (C®A mutation (4%, IVS II-654 (C®T (2%, codons 71/72 (+A (2% and codon 35 (C®A mutations (2% were also found. These techniques were found to be a valuable tool for analysis of b-thalassemia mutations because they are accurate, simple, and speedy in operation. The application for the diagnosis of severe thalassemia in high-risk pregnancies is promising.

  5. Rapid species specific identification and subtyping of Yersinia enterocolitica by MALDI-TOF mass spectrometry.

    Science.gov (United States)

    Stephan, Roger; Cernela, Nicole; Ziegler, Dominik; Pflüger, Valentin; Tonolla, Mauro; Ravasi, Damiana; Fredriksson-Ahomaa, Maria; Hächler, Herbert

    2011-11-01

    Yersinia enterocolitica are Gram-negative pathogens and known as important causes of foodborne infections. Rapid and reliable identification of strains of the species Y. enterocolitica within the genus Yersinia and the differentiation of the pathogenic from the non-pathogenic biotypes has become increasingly important. We evaluated here the application of matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS) for rapid species identification and subtyping of Y. enterocolitica. To this end, we developed a reference MS database library including 19 Y. enterocolitica (non-pathogenic biotype 1A and pathogenic biotypes 2 and 4) as well as 24 non-Y. enterocolitica strains, belonging to eleven different other Yersinia spp. The strains provided reproducible and unique mass spectra profiles covering a wide molecular mass range (2000 to 30,000 Da). Species-specific and biotype-specific biomarker protein mass patterns were determined for Y. enterocolitica. The defined biomarker mass patterns (SARAMIS SuperSpectrum™) were validated using 117 strains from various Y. enterocolitica bioserotypes in a blind-test. All strains were correctly identified and for all strains the mass spectrometry-based identification scheme yielded identical results compared to a characterization by a combination of biotyping and serotyping. Our study demonstrates that MALDI-TOF-MS is a reliable and powerful tool for the rapid identification of Y. enterocolitica strains to the species level and allows subtyping of strains to the biotype level. Copyright © 2011 Elsevier B.V. All rights reserved.

  6. Identification of somatic mutations in cancer through Bayesian-based analysis of sequenced genome pairs.

    Science.gov (United States)

    Christoforides, Alexis; Carpten, John D; Weiss, Glen J; Demeure, Michael J; Von Hoff, Daniel D; Craig, David W

    2013-05-04

    The field of cancer genomics has rapidly adopted next-generation sequencing (NGS) in order to study and characterize malignant tumors with unprecedented resolution. In particular for cancer, one is often trying to identify somatic mutations--changes specific to a tumor and not within an individual's germline. However, false positive and false negative detections often result from lack of sufficient variant evidence, contamination of the biopsy by stromal tissue, sequencing errors, and the erroneous classification of germline variation as tumor-specific. We have developed a generalized Bayesian analysis framework for matched tumor/normal samples with the purpose of identifying tumor-specific alterations such as single nucleotide mutations, small insertions/deletions, and structural variation. We describe our methodology, and discuss its application to other types of paired-tissue analysis such as the detection of loss of heterozygosity as well as allelic imbalance. We also demonstrate the high level of sensitivity and specificity in discovering simulated somatic mutations, for various combinations of a) genomic coverage and b) emulated heterogeneity. We present a Java-based implementation of our methods named Seurat, which is made available for free academic use. We have demonstrated and reported on the discovery of different types of somatic change by applying Seurat to an experimentally-derived cancer dataset using our methods; and have discussed considerations and practices regarding the accurate detection of somatic events in cancer genomes. Seurat is available at https://sites.google.com/site/seuratsomatic.

  7. [Evaluation of a rapid trehalase test for the identification of Candida glabrata].

    Science.gov (United States)

    Kirdar, Sevin; Gültekin, Berna; Evcil, Gonca; Ozkütük, Aydan; Sener, Asli Gamze; Aydin, Neriman

    2009-04-01

    Candida species which cause local infections, may also lead to fatal systemic infections. The increasing incidence of non-albicans Candida, especially fluconazole susceptible or resistant dose-dependent C. glabrata, increased the importance of rapid and accurate species level identification for Candida. Rapid and correct identification of C. glabrata is essential for the initiation of the appropriate antifungal therapy. This study was conducted to evaluate the performance of the rapid trehalase test in the diagnosis of C. glabrata isolates. A total of 173 Candida strains isolated from various clinical specimens and identified according to germ tube test, growth on cornmeal Tween 80 agar and the colony morphologies on Mast-CHROMagar Candida medium (Mast Diagnostics, UK), were included to the study. The identification of non-albicans Candida species were also confirmed by API 20CAUX (BioMerieux, France) system. Accordingly 86 (50%) of the isolates were identified as C. glabrata, 48 (28%) C. albicans, 17 (10%) C. krusei, 13 (8%) C. tropicalis, 5 (3%) C. parapsilosis, 3 (2%) C. kefyr and 1 (1%) Cutilis. In order to detect the presence of trehalase enzyme in Condida strains, all isolates were grown on Sabouraud dextrose agar containing 4% glucose and then one yeast colony was emulsified in 50 microl of citrate buffer containing 4% (wt/vol) trehalose for 3 h at 37 degrees C. Presence of glucose which emerged after the action of trehalase on trehalose, was detected by a commercial "urinary glucose detection dipstick" (Spinreacta, Spain). All C. glabrata strains yielded positive result by trehalase test. None C. glabrata isolates were found negative by trehalase test except for one strain of C. tropicalis. In this study, the trehalase test allowed identification of C. globrata with 100% sensitivity and 98.9% specificity. It was concluded that trehalase test is a rapid, cost-effective and simple test that can be used for the accurate identification of C. glabrata.

  8. Identification of the bovine Arachnomelia mutation by massively parallel sequencing implicates sulfite oxidase (SUOX in bone development.

    Directory of Open Access Journals (Sweden)

    Cord Drögemüller

    2010-08-01

    Full Text Available Arachnomelia is a monogenic recessive defect of skeletal development in cattle. The causative mutation was previously mapped to a ∼7 Mb interval on chromosome 5. Here we show that array-based sequence capture and massively parallel sequencing technology, combined with the typical family structure in livestock populations, facilitates the identification of the causative mutation. We re-sequenced the entire critical interval in a healthy partially inbred cow carrying one copy of the critical chromosome segment in its ancestral state and one copy of the same segment with the arachnomelia mutation, and we detected a single heterozygous position. The genetic makeup of several partially inbred cattle provides extremely strong support for the causality of this mutation. The mutation represents a single base insertion leading to a premature stop codon in the coding sequence of the SUOX gene and is perfectly associated with the arachnomelia phenotype. Our findings suggest an important role for sulfite oxidase in bone development.

  9. Identification of the Bovine Arachnomelia Mutation by Massively Parallel Sequencing Implicates Sulfite Oxidase (SUOX) in Bone Development

    Science.gov (United States)

    Drögemüller, Cord; Tetens, Jens; Sigurdsson, Snaevar; Gentile, Arcangelo; Testoni, Stefania; Lindblad-Toh, Kerstin; Leeb, Tosso

    2010-01-01

    Arachnomelia is a monogenic recessive defect of skeletal development in cattle. The causative mutation was previously mapped to a ∼7 Mb interval on chromosome 5. Here we show that array-based sequence capture and massively parallel sequencing technology, combined with the typical family structure in livestock populations, facilitates the identification of the causative mutation. We re-sequenced the entire critical interval in a healthy partially inbred cow carrying one copy of the critical chromosome segment in its ancestral state and one copy of the same segment with the arachnomelia mutation, and we detected a single heterozygous position. The genetic makeup of several partially inbred cattle provides extremely strong support for the causality of this mutation. The mutation represents a single base insertion leading to a premature stop codon in the coding sequence of the SUOX gene and is perfectly associated with the arachnomelia phenotype. Our findings suggest an important role for sulfite oxidase in bone development. PMID:20865119

  10. Rapid identification of Yersinia pestis and Brucella melitensis by chip-based continuous flow PCR

    Science.gov (United States)

    Dietzsch, Michael; Hlawatsch, Nadine; Melzer, Falk; Tomaso, Herbert; Gärtner, Claudia; Neubauer, Heinrich

    2012-06-01

    To combat the threat of biological agents like Yersinia pestis and Brucella melitensis in bioterroristic scenarios requires fast, easy-to-use and safe identification systems. In this study we describe a system for rapid amplification of specific genetic markers for the identification of Yersinia pestis and Brucella melitensis. Using chip based PCR and continuous flow technology we were able to amplify the targets simultaneously with a 2-step reaction profile within 20 minutes. The subsequent analysis of amplified fragments by standard gel electrophoresis requires another 45 minutes. We were able to detect both pathogens within 75 minutes being much faster than most other nucleic acid amplification technologies.

  11. Rapid detection of most frequent Slovenian germ-line mutations in BRCA1 gene using real-time PCR and melting curve analysis

    International Nuclear Information System (INIS)

    Novakovic, S.; Stegel, V.

    2005-01-01

    Background. Detection of inherited mutations in cancer susceptibility genes is of great importance in some types of cancers including the colorectal cancer (mutations of APC gene in familial adenomatous polyposis - FAP, mutations in mismatch repair genes in hereditary nonpolyposis colorectal cancer - HNPCC), malignant melanoma (mutations in CDKN2A and CDK4 genes) and breast cancer (mutations in BRCA1 and BRCA2 genes). Methods. This article presents the technical data for the detection of five mutations in BRCA1 gene in breast cancer patients and their relatives. The mutations - 1806C>T, 300T>G, 300T>A, 310G>A, 5382insC - were determined by the real-time PCR and the melting curve analysis. Results and conclusion. In comparison to direct sequencing, this method proved to be sensitive and rapid enough for the routine daily determination of mutations in DNA isolated from the peripheral blood. (author)

  12. Identification of novel BRCA founder mutations in Middle Eastern breast cancer patients using capture and Sanger sequencing analysis.

    Science.gov (United States)

    Bu, Rong; Siraj, Abdul K; Al-Obaisi, Khadija A S; Beg, Shaham; Al Hazmi, Mohsen; Ajarim, Dahish; Tulbah, Asma; Al-Dayel, Fouad; Al-Kuraya, Khawla S

    2016-09-01

    Ethnic differences of breast cancer genomics have prompted us to investigate the spectra of BRCA1 and BRCA2 mutations in different populations. The prevalence and effect of BRCA 1 and BRCA 2 mutations in Middle Eastern population is not fully explored. To characterize the prevalence of BRCA mutations in Middle Eastern breast cancer patients, BRCA mutation screening was performed in 818 unselected breast cancer patients using Capture and/or Sanger sequencing. 19 short tandem repeat (STR) markers were used for founder mutation analysis. In our study, nine different types of deleterious mutation were identified in 28 (3.4%) cases, 25 (89.3%) cases in BRCA 1 and 3 (10.7%) cases in BRCA 2. Seven recurrent mutations identified accounted for 92.9% (26/28) of all the mutant cases. Haplotype analysis was performed to confirm c.1140 dupG and c.4136_4137delCT mutations as novel putative founder mutation, accounting for 46.4% (13/28) of all BRCA mutant cases and 1.6% (13/818) of all the breast cancer cases, respectively. Moreover, BRCA 1 mutation was significantly associated with BRCA 1 protein expression loss (p = 0.0005). Our finding revealed that a substantial number of BRCA mutations were identified in clinically high risk breast cancer from Middle East region. Identification of the mutation spectrum, prevalence and founder effect in Middle Eastern population facilitates genetic counseling, risk assessment and development of cost-effective screening strategy. © 2016 UICC.

  13. Real-time PCR-based method for the rapid detection of extended RAS mutations using bridged nucleic acids in colorectal cancer.

    Science.gov (United States)

    Iida, Takao; Mizuno, Yukie; Kaizaki, Yasuharu

    2017-10-27

    Mutations in RAS and BRAF are predictors of the efficacy of anti-epidermal growth factor receptor (EGFR) therapy in patients with metastatic colorectal cancer (mCRC). Therefore, simple, rapid, cost-effective methods to detect these mutations in the clinical setting are greatly needed. In the present study, we evaluated BNA Real-time PCR Mutation Detection Kit Extended RAS (BNA Real-time PCR), a real-time PCR method that uses bridged nucleic acid clamping technology to rapidly detect mutations in RAS exons 2-4 and BRAF exon 15. Genomic DNA was extracted from 54 formalin-fixed paraffin-embedded (FFPE) tissue samples obtained from mCRC patients. Among the 54 FFPE samples, BNA Real-time PCR detected 21 RAS mutations (38.9%) and 5 BRAF mutations (9.3%), and the reference assay (KRAS Mutation Detection Kit and MEBGEN™ RASKET KIT) detected 22 RAS mutations (40.7%). The concordance rate of detected RAS mutations between the BNA Real-time PCR assay and the reference assays was 98.2% (53/54). The BNA Real-time PCR assay proved to be a more simple, rapid, and cost-effective method for detecting KRAS and RAS mutations compared with existing assays. These findings suggest that BNA Real-time PCR is a valuable tool for predicting the efficacy of early anti-EGFR therapy in mCRC patients. Copyright © 2017 Elsevier B.V. All rights reserved.

  14. Rapid identification of sequences for orphan enzymes to power accurate protein annotation.

    Directory of Open Access Journals (Sweden)

    Kevin R Ramkissoon

    Full Text Available The power of genome sequencing depends on the ability to understand what those genes and their proteins products actually do. The automated methods used to assign functions to putative proteins in newly sequenced organisms are limited by the size of our library of proteins with both known function and sequence. Unfortunately this library grows slowly, lagging well behind the rapid increase in novel protein sequences produced by modern genome sequencing methods. One potential source for rapidly expanding this functional library is the "back catalog" of enzymology--"orphan enzymes," those enzymes that have been characterized and yet lack any associated sequence. There are hundreds of orphan enzymes in the Enzyme Commission (EC database alone. In this study, we demonstrate how this orphan enzyme "back catalog" is a fertile source for rapidly advancing the state of protein annotation. Starting from three orphan enzyme samples, we applied mass-spectrometry based analysis and computational methods (including sequence similarity networks, sequence and structural alignments, and operon context analysis to rapidly identify the specific sequence for each orphan while avoiding the most time- and labor-intensive aspects of typical sequence identifications. We then used these three new sequences to more accurately predict the catalytic function of 385 previously uncharacterized or misannotated proteins. We expect that this kind of rapid sequence identification could be efficiently applied on a larger scale to make enzymology's "back catalog" another powerful tool to drive accurate genome annotation.

  15. Rapid Identification of Sequences for Orphan Enzymes to Power Accurate Protein Annotation

    Science.gov (United States)

    Ojha, Sunil; Watson, Douglas S.; Bomar, Martha G.; Galande, Amit K.; Shearer, Alexander G.

    2013-01-01

    The power of genome sequencing depends on the ability to understand what those genes and their proteins products actually do. The automated methods used to assign functions to putative proteins in newly sequenced organisms are limited by the size of our library of proteins with both known function and sequence. Unfortunately this library grows slowly, lagging well behind the rapid increase in novel protein sequences produced by modern genome sequencing methods. One potential source for rapidly expanding this functional library is the “back catalog” of enzymology – “orphan enzymes,” those enzymes that have been characterized and yet lack any associated sequence. There are hundreds of orphan enzymes in the Enzyme Commission (EC) database alone. In this study, we demonstrate how this orphan enzyme “back catalog” is a fertile source for rapidly advancing the state of protein annotation. Starting from three orphan enzyme samples, we applied mass-spectrometry based analysis and computational methods (including sequence similarity networks, sequence and structural alignments, and operon context analysis) to rapidly identify the specific sequence for each orphan while avoiding the most time- and labor-intensive aspects of typical sequence identifications. We then used these three new sequences to more accurately predict the catalytic function of 385 previously uncharacterized or misannotated proteins. We expect that this kind of rapid sequence identification could be efficiently applied on a larger scale to make enzymology’s “back catalog” another powerful tool to drive accurate genome annotation. PMID:24386392

  16. Performance of chromogenic media for Candida in rapid presumptive identification of Candida species from clinical materials

    OpenAIRE

    Pravin Charles, M. V.; Kali, Arunava; Joseph, Noyal Mariya

    2015-01-01

    Background: In perspective of the worldwide increase in a number of immunocompromised patients, the need for identification of Candida species has become a major concern. The development of chromogenic differential media, introduced recently, facilitate rapid speciation. However, it can be employed for routine mycology workup only after an exhaustive evaluation of its benefit and cost effectiveness. This study was undertaken to evaluate the benefit and cost effectiveness of chromogenic media ...

  17. Molecular genetics of the Usher syndrome in Lebanon: identification of 11 novel protein truncating mutations by whole exome sequencing.

    Science.gov (United States)

    Reddy, Ramesh; Fahiminiya, Somayyeh; El Zir, Elie; Mansour, Ahmad; Megarbane, Andre; Majewski, Jacek; Slim, Rima

    2014-01-01

    Usher syndrome (USH) is a genetically heterogeneous condition with ten disease-causing genes. The spectrum of genes and mutations causing USH in the Lebanese and Middle Eastern populations has not been described. Consequently, diagnostic approaches designed to screen for previously reported mutations were unlikely to identify the mutations in 11 unrelated families, eight of Lebanese and three of Middle Eastern origins. In addition, six of the ten USH genes consist of more than 20 exons, each, which made mutational analysis by Sanger sequencing of PCR-amplified exons from genomic DNA tedious and costly. The study was aimed at the identification of USH causing genes and mutations in 11 unrelated families with USH type I or II. Whole exome sequencing followed by expanded familial validation by Sanger sequencing. We identified disease-causing mutations in all the analyzed patients in four USH genes, MYO7A, USH2A, GPR98 and CDH23. Eleven of the mutations were novel and protein truncating, including a complex rearrangement in GPR98. Our data highlight the genetic diversity of Usher syndrome in the Lebanese population and the time and cost-effectiveness of whole exome sequencing approach for mutation analysis of genetically heterogeneous conditions caused by large genes.

  18. Molecular genetics of the Usher syndrome in Lebanon: identification of 11 novel protein truncating mutations by whole exome sequencing.

    Directory of Open Access Journals (Sweden)

    Ramesh Reddy

    Full Text Available Usher syndrome (USH is a genetically heterogeneous condition with ten disease-causing genes. The spectrum of genes and mutations causing USH in the Lebanese and Middle Eastern populations has not been described. Consequently, diagnostic approaches designed to screen for previously reported mutations were unlikely to identify the mutations in 11 unrelated families, eight of Lebanese and three of Middle Eastern origins. In addition, six of the ten USH genes consist of more than 20 exons, each, which made mutational analysis by Sanger sequencing of PCR-amplified exons from genomic DNA tedious and costly. The study was aimed at the identification of USH causing genes and mutations in 11 unrelated families with USH type I or II.Whole exome sequencing followed by expanded familial validation by Sanger sequencing.We identified disease-causing mutations in all the analyzed patients in four USH genes, MYO7A, USH2A, GPR98 and CDH23. Eleven of the mutations were novel and protein truncating, including a complex rearrangement in GPR98.Our data highlight the genetic diversity of Usher syndrome in the Lebanese population and the time and cost-effectiveness of whole exome sequencing approach for mutation analysis of genetically heterogeneous conditions caused by large genes.

  19. Molecular Genetics of the Usher Syndrome in Lebanon: Identification of 11 Novel Protein Truncating Mutations by Whole Exome Sequencing

    Science.gov (United States)

    Reddy, Ramesh; Fahiminiya, Somayyeh; El Zir, Elie; Mansour, Ahmad; Megarbane, Andre; Majewski, Jacek; Slim, Rima

    2014-01-01

    Background Usher syndrome (USH) is a genetically heterogeneous condition with ten disease-causing genes. The spectrum of genes and mutations causing USH in the Lebanese and Middle Eastern populations has not been described. Consequently, diagnostic approaches designed to screen for previously reported mutations were unlikely to identify the mutations in 11 unrelated families, eight of Lebanese and three of Middle Eastern origins. In addition, six of the ten USH genes consist of more than 20 exons, each, which made mutational analysis by Sanger sequencing of PCR-amplified exons from genomic DNA tedious and costly. The study was aimed at the identification of USH causing genes and mutations in 11 unrelated families with USH type I or II. Methods Whole exome sequencing followed by expanded familial validation by Sanger sequencing. Results We identified disease-causing mutations in all the analyzed patients in four USH genes, MYO7A, USH2A, GPR98 and CDH23. Eleven of the mutations were novel and protein truncating, including a complex rearrangement in GPR98. Conclusion Our data highlight the genetic diversity of Usher syndrome in the Lebanese population and the time and cost-effectiveness of whole exome sequencing approach for mutation analysis of genetically heterogeneous conditions caused by large genes. PMID:25211151

  20. Mutational analysis of GALT gene in Greek patients with galactosaemia: identification of two novel mutations and clinical evaluation.

    Science.gov (United States)

    Schulpis, Kleopatra H; Thodi, Georgia; Iakovou, Konstantinos; Chatzidaki, Maria; Dotsikas, Yannis; Molou, Elina; Triantafylli, Olga; Loukas, Yannis L

    2017-10-01

    Classical galactosaemia is an inborn error of metabolism due to the deficiency of the enzyme galactose-1-phosphate uridylyltransferase (GALT). The aim of the study was to identify the underlying mutations in Greek patients with GALT deficiency and evaluate their psychomotor and speech development. Patients with GALT deficiency (n = 17) were picked up through neonatal screening. Mutational analysis was conducted via Sanger sequencing, while in silico analysis was used in the cases of novel missense mutations. Psychomotor speech development tests were utilized for the clinical evaluation of the patients. Eleven different mutations in the GALT gene were detected in the patient cohort, including two novel ones. The most frequent mutation was p.Q188R (c.563 A > G). As for the novel mutations, p.M298I (c.894 G > A) was identified in four out of 32 independent alleles, while p.P115S (c.343 C > T) was identified once. Psychomotor evaluation revealed that most of the patients were found in the borderline area (Peabody test), while only two had speech delay problems. The WISK test revealed three patients at borderline limits and two were at lower than normal limits. The mutational spectrum of the GALT gene in Greek patients is presented for the first time. The mutation p.Q188R is the most frequent among Greek patients. Two novel mutations were identified and their potential pathogenicity was estimated. Regarding the phenotypic characteristics, psychomotor disturbances and speech delay were mainly observed among GALT-deficient patients.

  1. A rapid identification of four medicinal chrysanthemum varieties with near infrared spectroscopy.

    Science.gov (United States)

    Han, Bangxing; Yan, Hui; Chen, Cunwu; Yao, Houjun; Dai, Jun; Chen, Naifu

    2014-07-01

    For genuine medicinal material in Chinese herbs; the efficient, rapid, and precise identification is the focus and difficulty in the filed studying Chinese herbal medicines. Chrysanthemum morifolium as herbs has a long planting history in China, culturing high quality ones and different varieties. Different chrysanthemum varieties differ in quality, chemical composition, functions, and application. Therefore, chrysanthemum varieties in the market demands precise identification to provide reference for reasonable and correct application as genuine medicinal material. A total of 244 batches of chrysanthemum samples were randomly divided into calibration set (160 batches) and prediction set (84 batches). The near infrared diffuses reflectance spectra of chrysanthemum varieties were preprocessed by first order derivative (D1) and autoscaling and was built model with partial least squares (PLS). In this study of four chrysanthemum varieties identification, the accuracy rates in calibration sets of Boju, Chuju, Hangju, and Gongju are respectively 100, 100, 98.65, and 96.67%; while the accuracy rates in prediction sets are 100% except for 99.1% of Hangju. The research results demonstrate that the qualitative analysis can be conducted by machine learning combined with near infrared spectroscopy (NIR), which provides a new method for rapid and noninvasive identification of chrysanthemum varieties.

  2. MALDI-TOF mass spectrometry for the rapid identification of aetiological agents of sepsis

    Directory of Open Access Journals (Sweden)

    Roberto Degl’Innocenti

    2013-04-01

    Full Text Available Introduction: The MALDI-TOF has recently become part of the methods of microbiological investigation in many laboratories of bacteriology with advantages both practical and economical.The use of this technique for the rapid identification of the causative agents of sepsis is of strategic importance to the ability to provide the clinician with useful information for a prompt and rapid establishment of an empirical antimicrobial “targeted” therapy. Methods: It was tested a total of 343 positive blood culture bottles from 211 patients. The samples after collection were incubated in the BACTEC FX (Becton Dickinson, USA. From these bottles were taken a few milliliters of broth culture and transferred into a vacutainer tube containing gel. This was centrifuged, the supernatant was decanted, and finally recovered the bacterial suspension on the gel. With micro-organisms recovered in this way, after several washes with distilled water, was prepared a slide for microscopic examination with Gram stain, and a plate for mass spectrometry (MS-Vitek, bioMérieux, France.Then, the same samples were inoculated on solid agar media according to the protocol in use in our laboratory.The next day was checked the possible bacterial growth on solid media; we then proceeded to the identification of the colonies by Vitek MS and / or with the system Vitek2 (bioMérieux, France. Results: 258 (75.2% positive vials show concordant results between direct identification and identification after growth on agar. For 83 (24.2% positive bottles there has been full compliance with the microscopic examination but not with culture. In particular, two bottles (0.6% have given complete discordance between the direct identification and that after growth. Conclusions: The protocol we use for the direct identification of organisms responsible for sepsis, directly on positive bottles, seems to be a quick and inexpensive procedure, which in less than 60 minutes can give valuable

  3. Rapid identification of herbal compounds derived metabolites using zebrafish larvae as the biotransformation system.

    Science.gov (United States)

    Wang, Chen; Yin, Ying-Hao; Wei, Ying-Jie; Shi, Zi-Qi; Liu, Jian-Qun; Liu, Li-Fang; Xin, Gui-Zhong

    2017-09-15

    Metabolites derived from herbal compounds are becoming promising sources for discovering new drugs. However, the rapid identification of metabolites from biological matrixes is limited by massive endogenous interference and low abundance of metabolites. Thus, by using zebrafish larvae as the biotransformation system, we herein proposed and validated an integrated strategy for rapid identification of metabolites derived from herbal compounds. Two pivotal steps involved in this strategy are to differentiate metabolites from herbal compounds and match metabolites with their parent compounds. The differentiation step was achieved by cross orthogonal partial least-squares discriminant analysis. Automatic matching analysis was performed on R Project based on a self-developed program, of which the number of matched ionic clusters and its corresponding percentage between metabolite and parent compound were taken into account to assess their similarity. Using this strategy, 46 metabolites screened from incubation water samples of zebrafish treated with total Epimedium flavonoids (EFs) could be matched with their corresponding parent compounds, 37 of them were identified and validated by the known metabolic pathways and fragmentation patterns. Finally, 75% of the identified EFs metabolites were successfully detected in urine samples of rats treated with EFs. These experimental results indicate that the proposed strategy using zebrafish larvae as the biotransformation system will facilitate the rapid identification of metabolites derived from herbal compounds, which shows promising perspectives in providing additional resources for pharmaceutical developments from natural products. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. Identification of a two base pair deletion in five unrelated families with adrenoleukodystrophy: a possible hot spot for mutations

    NARCIS (Netherlands)

    Kemp, S.; Ligtenberg, M. J.; van Geel, B. M.; Barth, P. G.; Wolterman, R. A.; Schoute, F.; Sarde, C. O.; Mandel, J. L.; van Oost, B. A.; Bolhuis, P. A.

    1994-01-01

    The gene for X-linked adrenoleukodystrophy (ALD) was recently identified. Intragenic deletions of several kilobases were found in about 7% of patients. Point mutations, expected to be very heterogeneous, were identified so far in only two patients. We report the identification of a two base pair

  5. Next-Gen Sequencing-Based Mapping and Identification of Ethyl Methanesulfonate-Induced Mutations in Arabidopsis thaliana.

    Science.gov (United States)

    Zhang, Xue-Cheng; Millet, Yves; Ausubel, Frederick M; Borowsky, Mark

    2014-10-01

    Forward genetic analysis using ethyl methanesulfonate (EMS) mutagenesis has proven to be a powerful tool in biological research, but identification and cloning of causal mutations by conventional genetic mapping approaches is a painstaking process. Recent advances in next-gen sequencing have greatly invigorated the process of identifying EMS-induced mutations corresponding to a specific phenotype in model genetic hosts, including the plant Arabidopsis thaliana and the nematode Caenorhabditis elegans. Next-gen sequencing of bulked F2 mutant recombinants produces a wealth of high-resolution genetic data, provides enhanced delimitation of the genomic location of mutations, and greatly reduces hands-on time while maintaining high accuracy and reproducibility. In this unit, a detailed procedure to simultaneously map and identify EMS mutations in Arabidopsis is described. Copyright © 2014 John Wiley & Sons, Inc.

  6. Rapid screening of β-Globin gene mutations by Real-Time PCR in ...

    African Journals Online (AJOL)

    Introduction of the real time PCR has made a revolution in the time taken for the PCR reactions. We present a method for the diagnosis of the common mutations of the B-thalassemia in Egyptian children & families. The procedure depends on the real-time PCR using specific fluorescently labeled hybridization probes.

  7. Toward Automating HIV Identification: Machine Learning for Rapid Identification of HIV-Related Social Media Data.

    Science.gov (United States)

    Young, Sean D; Yu, Wenchao; Wang, Wei

    2017-02-01

    "Social big data" from technologies such as social media, wearable devices, and online searches continue to grow and can be used as tools for HIV research. Although researchers can uncover patterns and insights associated with HIV trends and transmission, the review process is time consuming and resource intensive. Machine learning methods derived from computer science might be used to assist HIV domain experts by learning how to rapidly and accurately identify patterns associated with HIV from a large set of social data. Using an existing social media data set that was associated with HIV and coded by an HIV domain expert, we tested whether 4 commonly used machine learning methods could learn the patterns associated with HIV risk behavior. We used the 10-fold cross-validation method to examine the speed and accuracy of these models in applying that knowledge to detect HIV content in social media data. Logistic regression and random forest resulted in the highest accuracy in detecting HIV-related social data (85.3%), whereas the Ridge Regression Classifier resulted in the lowest accuracy. Logistic regression yielded the fastest processing time (16.98 seconds). Machine learning can enable social big data to become a new and important tool in HIV research, helping to create a new field of "digital HIV epidemiology." If a domain expert can identify patterns in social data associated with HIV risk or HIV transmission, machine learning models could quickly and accurately learn those associations and identify potential HIV patterns in large social data sets.

  8. Rapid identification of Enterobacter hormaechei and Enterobacter cloacae genetic cluster III.

    Science.gov (United States)

    Ohad, S; Block, C; Kravitz, V; Farber, A; Pilo, S; Breuer, R; Rorman, E

    2014-05-01

    Enterobacter cloacae complex bacteria are of both clinical and environmental importance. Phenotypic methods are unable to distinguish between some of the species in this complex, which often renders their identification incomplete. The goal of this study was to develop molecular assays to identify Enterobacter hormaechei and Ent. cloacae genetic cluster III which are relatively frequently encountered in clinical material. The molecular assays developed in this study are qPCR technology based and served to identify both Ent. hormaechei and Ent. cloacae genetic cluster III. qPCR results were compared to hsp60 sequence analysis. Most clinical isolates were assigned to Ent. hormaechei subsp. steigerwaltii and Ent. cloacae genetic cluster III. The latter was proportionately more frequently isolated from bloodstream infections than from other material (P < 0·05). The qPCR assays detecting Ent. hormaechei and Ent. cloacae genetic cluster III demonstrated high sensitivity and specificity. The presented qPCR assays allow accurate and rapid identification of clinical isolates of the Ent. cloacae complex. The improved identifications obtained can specifically assist analysis of Ent. hormaechei and Ent. cloacae genetic cluster III in nosocomial outbreaks and can promote rapid environmental monitoring. An association was observed between Ent. cloacae cluster III and systemic infection that deserves further attention. © 2014 The Society for Applied Microbiology.

  9. Final Progress Report: Isotope Identification Algorithm for Rapid and Accurate Determination of Radioisotopes Feasibility Study

    International Nuclear Information System (INIS)

    Rawool-Sullivan, Mohini; Bounds, John Alan; Brumby, Steven P.; Prasad, Lakshman; Sullivan, John P.

    2012-01-01

    This is the final report of the project titled, 'Isotope Identification Algorithm for Rapid and Accurate Determination of Radioisotopes,' PMIS project number LA10-HUMANID-PD03. The goal of the work was to demonstrate principles of emulating a human analysis approach towards the data collected using radiation isotope identification devices (RIIDs). It summarizes work performed over the FY10 time period. The goal of the work was to demonstrate principles of emulating a human analysis approach towards the data collected using radiation isotope identification devices (RIIDs). Human analysts begin analyzing a spectrum based on features in the spectrum - lines and shapes that are present in a given spectrum. The proposed work was to carry out a feasibility study that will pick out all gamma ray peaks and other features such as Compton edges, bremsstrahlung, presence/absence of shielding and presence of neutrons and escape peaks. Ultimately success of this feasibility study will allow us to collectively explain identified features and form a realistic scenario that produced a given spectrum in the future. We wanted to develop and demonstrate machine learning algorithms that will qualitatively enhance the automated identification capabilities of portable radiological sensors that are currently being used in the field.

  10. Rapid experimental SAD phasing and hot-spot identification with halogenated fragments

    Directory of Open Access Journals (Sweden)

    Joseph D. Bauman

    2016-01-01

    Full Text Available Through X-ray crystallographic fragment screening, 4-bromopyrazole was discovered to be a `magic bullet' that is capable of binding at many of the ligand `hot spots' found in HIV-1 reverse transcriptase (RT. The binding locations can be in pockets that are `hidden' in the unliganded crystal form, allowing rapid identification of these sites for in silico screening. In addition to hot-spot identification, this ubiquitous yet specific binding provides an avenue for X-ray crystallographic phase determination, which can be a significant bottleneck in the determination of the structures of novel proteins. The anomalous signal from 4-bromopyrazole or 4-iodopyrazole was sufficient to determine the structures of three proteins (HIV-1 RT, influenza A endonuclease and proteinase K by single-wavelength anomalous dispersion (SAD from single crystals. Both compounds are inexpensive, readily available, safe and very soluble in DMSO or water, allowing efficient soaking into crystals.

  11. Can the rapid identification of mature spermatozoa during microdissection testicular sperm extraction guide operative planning?

    Science.gov (United States)

    Alrabeeah, K; Doucet, R; Boulet, E; Phillips, S; Al-Hathal, N; Bissonnette, F; Kadoch, I J; Zini, A

    2015-05-01

    The minimum sperm count and quality that must be identified during microdissection testicular sperm extraction (micro-TESE) to deem the procedure successful remains to be established. We conducted a retrospective study of 81 consecutive men with non-obstructive azoospermia who underwent a primary (first) micro-TESE between March 2007 and October 2013. Final assessment of sperm recovery [reported on the day of (intracytoplasmic sperm injection) ICSI] was recorded as (i) successful (available spermatozoa for ICSI) or (ii) unsuccessful (no spermatozoa for ICSI). The decision to perform a unilateral (with limited or complete microdissection) or bilateral micro-TESE was guided by the intra-operative identification of sperm recovery (≥5 motile or non-motile sperm) from the first testicle. Overall, sperm recovery was successful in 56% (45/81) of the men. A unilateral micro-TESE was performed in 47% (38/81) of the men (based on intra-operative identification of sperm) and in 100% (38/38) of these men, spermatozoa was found on final assessment. In 42% (16/38) of the unilateral cases, a limited microdissection was performed (owing to the rapid intra-operative identification of sperm). The remaining 43 men underwent a bilateral micro-TESE and 16% (7/43) of these men had sperm identified on final assessment. The cumulative ICSI pregnancy rates (per cycle started and per embryo transfer) were 47% (21/45) and 60% (21/35), respectively, with a mean (±SD) of 1.9 ± 1.0 embryos transferred. The data demonstrate that intra-operative assessment of sperm recovery can correctly identify those men that require a unilateral micro-TESE. Moreover, the rapid identification of sperm recovery can allow some men to undergo a limited unilateral micro-TESE and avoid the need for complete testicular microdissection. © 2015 American Society of Andrology and European Academy of Andrology.

  12. Rapid identification of the Asian gypsy moth and its related species based on mitochondrial DNA.

    Science.gov (United States)

    Wu, Ying; Du, Qiuyang; Qin, Haiwen; Shi, Juan; Wu, Zhiyi; Shao, Weidong

    2018-02-01

    The gypsy moth- Lymantria dispar (Linnaeus)-is a worldwide forest defoliator and is of two types: the European gypsy moth and the Asian gypsy moth. Because of multiple invasions of the Asian gypsy moth, the North American Plant Protection Organization officially approved Regional Standards for Phytosanitary Measures No. 33. Accordingly, special quarantine measures have been implemented for 30 special focused ports in the epidemic areas of the Asian gypsy moth, including China, which has imposed great inconvenience on export trade. The Asian gypsy moth and its related species (i.e., Lymantria monocha and Lymantria xylina ) intercepted at ports are usually at different life stages, making their identification difficult. Furthermore, Port quarantine requires speedy clearance. As such, it is difficult to identify the Asian gypsy moth and its related species only by their morphological characteristics in a speedy measure. Therefore, this study aimed to use molecular biology technology to rapidly identify the Asian gypsy moth and its related species based on the consistency of mitochondrial DNA in different life stages. We designed 10 pairs of specific primers from different fragments of the Asian gypsy moth and its related species, and their detection sensitivity met the need for rapid identification. In addition, we determined the optimal polymerase chain reaction amplification temperature of the 10 pairs of specific primers, including three pairs of specific primers for the Asian gypsy moth ( L. dispar asiatic ), four pairs of specific primers for the nun moth ( L. monocha ), and three pairs of specific primers for the casuarina moth ( L. xylina ). In conclusion, using our designed primers, direct rapid identification of the Asian gypsy moth and its related species is possible, and this advancement can help improve export trade in China.

  13. Validation of Ion TorrentTM Inherited Disease Panel with the PGMTM Sequencing Platform for Rapid and Comprehensive Mutation Detection

    Directory of Open Access Journals (Sweden)

    Abeer E. Mustafa

    2018-05-01

    Full Text Available Quick and accurate molecular testing is necessary for the better management of many inherited diseases. Recent technological advances in various next generation sequencing (NGS platforms, such as target panel-based sequencing, has enabled comprehensive, quick, and precise interrogation of many genetic variations. As a result, these technologies have become a valuable tool for gene discovery and for clinical diagnostics. The AmpliSeq Inherited Disease Panel (IDP consists of 328 genes underlying more than 700 inherited diseases. Here, we aimed to assess the performance of the IDP as a sensitive and rapid comprehensive gene panel testing. A total of 88 patients with inherited diseases and causal mutations that were previously identified by Sanger sequencing were randomly selected for assessing the performance of the IDP. The IDP successfully detected 93.1% of the mutations in our validation cohort, achieving high overall gene coverage (98%. The sensitivity for detecting single nucleotide variants (SNVs and short Indels was 97.3% and 69.2%, respectively. IDP, when coupled with Ion Torrent Personal Genome Machine (PGM, delivers comprehensive and rapid sequencing for genes that are responsible for various inherited diseases. Our validation results suggest the suitability of this panel for use as a first-line screening test after applying the necessary clinical validation.

  14. Toward Male Individualization with Rapidly Mutating Y-Chromosomal Short Tandem Repeats

    NARCIS (Netherlands)

    K. Ballantyne (Kaye); A. Ralf (Arwin); R. Aboukhalid (Rachid); N.M. Achakzai (Niaz); T. Anjos (Tania); Q. Ayub (Qasim); J. Balažic (Jože); J. Ballantyne (Jack); D.J. Ballard (David); B. Berger (Burkhard); C. Bobillo (Cecilia); M. Bouabdellah (Mehdi); H. Burri (Helen); T. Capal (Tomas); S. Caratti (Stefano); J. Cárdenas (Jorge); F. Cartault (François); E.F. Carvalho (Elizeu); M. de Carvalho (Margarete); B. Cheng (Baowen); M.D. Coble (Michael); D. Comas (David); D. Corach (Daniel); M. D'Amato (Mauro); S. Davison (Sean); P. de Knijff (Peter); M.C.A. de Ungria (Maria Corazon); R. Decorte (Ronny); T. Dobosz (Tadeusz); B.M. Dupuy (Berit); S. Elmrghni (Samir); M. Gliwiński (Mateusz); S.C. Gomes (Sara); L. Grol (Laurens); C. Haas (Cordula); E. Hanson (Erin); J. Henke (Jürgen); L. Henke (Lotte); F. Herrera-Rodríguez (Fabiola); C.R. Hill (Carolyn); G. Holmlund (Gunilla); K. Honda (Katsuya); U.-D. Immel (Uta-Dorothee); S. Inokuchi (Shota); R. Jobling; M. Kaddura (Mahmoud); J.S. Kim (Jong); S.H. Kim (Soon); W. Kim (Wook); T.E. King (Turi); E. Klausriegler (Eva); D. Kling (Daniel); L. Kovačević (Lejla); L. Kovatsi (Leda); P. Krajewski (Paweł); S. Kravchenko (Sergey); M.H.D. Larmuseau (Maarten); E.Y. Lee (Eun Young); R. Lessig (Rüdiger); L.A. Livshits (Ludmila); D. Marjanović (Damir); M. Minarik (Marek); N. Mizuno (Natsuko); H. Moreira (Helena); N. Morling (Niels); M. Mukherjee (Meeta); P. Munier (Patrick); J. Nagaraju (Javaregowda); F. Neuhuber (Franz); S. Nie (Shengjie); P. Nilasitsataporn (Premlaphat); T. Nishi (Takeki); H.H. Oh (Hye); S. Olofsson (Sylvia); V. Onofri (Valerio); J. Palo (Jukka); H. Pamjav (Horolma); W. Parson (Walther); M. Petlach (Michal); C. Phillips (Christopher); R. Ploski (Rafal); S.P.R. Prasad (Samayamantri P.); D. Primorac (Dragan); G.A. Purnomo (Gludhug); J. Purps (Josephine); H. Rangel-Villalobos (Hector); K. Reogonekbała (Krzysztof); B. Rerkamnuaychoke (Budsaba); D.R. Gonzalez (Danel Rey); C. Robino (Carlo); L. Roewer (Lutz); A. de Rosa (Anna); A. Sajantila (Antti); A. Sala (Andrea); J.M. Salvador (Jazelyn); P. Sanz (Paula); C. Schmitt (Christian); A.K. Sharma (Anisha K.); D.A. Silva (Dayse); K.-J. Shin (Kyoung-Jin); T. Sijen (Titia); M. Sirker (Miriam); D. Siváková (Daniela); V. Škaro (Vedrana); C. Solano-Matamoros (Carlos); L. Souto (L.); V. Stenzl (Vlastimil); H. Sudoyo (Herawati); D. Syndercombe-Court (Denise); A. Tagliabracci (Adriano); D. Taylor (Duncan); A. Tillmar (Andreas); I.S. Tsybovsky (Iosif); C. Tyler-Smith (Chris); K. van der Gaag (Kristiaan); D. Vanek (Daniel); A. Völgyi (Antónia); D. Ward (Denise); P. Willemse (Patricia); E.P.H. Yap (Eric); Z-Y. Yong (Ze-Yie); I.Z. Pajnič (Irena Zupanič); M.H. Kayser (Manfred)

    2014-01-01

    textabstractRelevant for various areas of human genetics, Y-chromosomal short tandem repeats (Y-STRs) are commonly used for testing close paternal relationships among individuals and populations, and for male lineage identification. However, even the widely used 17-loci Yfiler set cannot resolve

  15. A rapid MALDI-TOF MS identification database at genospecies level for clinical and environmental Aeromonas strains.

    Directory of Open Access Journals (Sweden)

    Cinzia Benagli

    Full Text Available The genus Aeromonas has undergone a number of taxonomic and nomenclature revisions over the past 20 years, and new (subspecies and biogroups are continuously described. Standard identification methods such as biochemical characterization have deficiencies and do not allow clarification of the taxonomic position. This report describes the development of a matrix-assisted laser desorption/ionisation-time of flight mass spectrometry (MALDI-TOF MS identification database for a rapid identification of clinical and environmental Aeromonas isolates.

  16. The rapid identification of human influenza neuraminidase N1 and N2 subtypes by ELISA.

    Science.gov (United States)

    Barr, I G; McCaig, M; Durrant, C; Shaw, R

    2006-11-10

    An ELISA assay was developed to allow the rapid and accurate identification of human influenza A N1 and N2 neuraminidases. Initial testing using a fetuin pre-coating of wells correctly identified 81.7% of the neuraminidase type from a series of human A(H1N1), A(H1N2) and A(H3N2) viruses. This result could be improved to detect the neuraminidase subtype of almost all human influenza A viruses from a large panel of viruses isolated from 2000 to 2005, if the fetuin pre-coating was removed and the viruses were coated directly onto wells. This method is simple, rapid and can be used to screen large numbers of currently circulating human influenza A viruses for their neurraminidase subtype and is a good alternative to RT-PCR.

  17. Identification of new adventitious rooting mutants amongst suppressors of the Arabidopsis thaliana superroot2 mutation.

    Science.gov (United States)

    Pacurar, Daniel Ioan; Pacurar, Monica Lacramioara; Bussell, John Desmond; Schwambach, Joseli; Pop, Tiberia Ioana; Kowalczyk, Mariusz; Gutierrez, Laurent; Cavel, Emilie; Chaabouni, Salma; Ljung, Karin; Fett-Neto, Arthur Germano; Pamfil, Doru; Bellini, Catherine

    2014-04-01

    The plant hormone auxin plays a central role in adventitious rooting and is routinely used with many economically important, vegetatively propagated plant species to promote adventitious root initiation and development on cuttings. Nevertheless the molecular mechanisms through which it acts are only starting to emerge. The Arabidopsis superroot2-1 (sur2-1) mutant overproduces auxin and, as a consequence, develops excessive adventitious roots in the hypocotyl. In order to increase the knowledge of adventitious rooting and of auxin signalling pathways and crosstalk, this study performed a screen for suppressors of superroot2-1 phenotype. These suppressors provide a new resource for discovery of genetic players involved in auxin signalling pathways or at the crosstalk of auxin and other hormones or environmental signals. This study reports the identification and characterization of 26 sur2-1 suppressor mutants, several of which were identified as mutations in candidate genes involved in either auxin biosynthesis or signalling. In addition to confirming the role of auxin as a central regulator of adventitious rooting, superroot2 suppressors indicated possible crosstalk with ethylene signalling in this process.

  18. Mutational analysis in patients with Autosomal Dominant Polycystic Kidney Disease (ADPKD): Identification of five mutations in the PKD1 gene.

    Science.gov (United States)

    Abdelwahed, Mayssa; Hilbert, Pascale; Ahmed, Asma; Mahfoudh, Hichem; Bouomrani, Salem; Dey, Mouna; Hachicha, Jamil; Kamoun, Hassen; Keskes-Ammar, Leila; Belguith, Neïla

    2018-05-31

    Autosomal Dominant Polycystic Kidney Disease (ADPKD), the most frequent genetic disorder of the kidneys, is characterized by a typical presenting symptoms include cysts development in different organs and a non-cysts manifestations. ADPKD is caused by mutations in PKD1 or PKD2 genes. In this study, we aimed to search for molecular causative defects among PKD1 and PKD2 genes. Eighteen patients were diagnosed based on renal ultrasonography and renal/extra-renal manifestations. Then, Sanger sequencing was performed for PKD1 and PKD2 genes. Multiplex Ligation dependent Probe Amplification method (MLPA) methods was performed for both PKD genes. Mutational analysis of the PKD2 gene revealed the absence of variants and no deletions or duplications of both PKD genes were detected. But three novels mutations i.e. p.S463C exon 7; c. c.11156+2T>C IVS38 and c.8161-1G>A IVS22 and two previously reported c.1522T>C exon 7 and c.412C>T exon 4 mutations in the PKD1 gene were detected. Bioinformatics tools predicted that the novel variants have a pathogenic effects on splicing machinery, pre-mRNA secondary structure and stability and protein stability. Our results highlighted molecular features of Tunisian patients with ADPKD and revealed novel variations that can be utilized in clinical diagnosis and in the evaluation of living kidney donor. To the best of our knowledge, this is the first report of Autosomal Polycystic Kidney Disease in Tunisia. Copyright © 2017. Published by Elsevier B.V.

  19. Identification of a novel p.R1443W mutation in RP1 gene associated with retinitis pigmentosa sine pigmento

    Directory of Open Access Journals (Sweden)

    Li Ma

    2013-08-01

    Full Text Available AIM: To screen mutations in the retinitis pigmentosa 1 (RP1 gene and the rhodopsin (RHO gene in Chinese patients with retinitis pigmentosa sine pigmento (RPSP and describe the genotype-phenotype relationship of the mutations.METHODS:Twenty affected, unrelated Chinese individuals with RPSP (4 autosomal dominant RPSP, 12 autosomal recessive RPSP and 4 unknown inheritance pattern were recruited between 2009 and 2012. The clinical features were determined by complete ophthalmologic examinations. Polymerase chain reaction (PCR and direct DNA sequencing were used to screen the entire coding region and splice junctions of the RP1 gene and the RHO gene. The cosegregation analysis and population frequency studies were performed for patients with identified mutations.RESULTS: Five variants in the RP1 gene and one in the RHO gene were detected in 20 probands. Four missense changes (rs444772, rs446227, rs414352, rs441800 and one non-coding variant (rs56340615 were common SNPs and none of them showed a significant relationship with RPSP. A missense mutation p.R1443W was identified in the RP1 gene in three affected individuals from a family with autosomal dominant RPSP and was found to cosegregate with the phenotype in this family, suggestive of pathogenic. In addition, population frequency analysis showed the p.R1443W mutation was absent in 300 healthy controls.CONCLUSION: The identification of p.R1443W mutation cosegregating in a family with autosomal dominant RPSP highlights an atypical phenotype of the RP1 gene mutation, while RHO gene is not associated with the pathogenesis of RPSP in this study. To our knowledge, this is the fist mutation identified to associate with RPSP.

  20. Identification of a novel p.R1443W mutation in RP1 gene associated with retinitis pigmentosa sine pigmento.

    Science.gov (United States)

    Ma, Li; Sheng, Xun-Lun; Li, Hui-Ping; Zhang, Fang-Xia; Liu, Ya-Ni; Rong, Wei-Ning; Zhang, Jian-Ling

    2013-01-01

    To screen mutations in the retinitis pigmentosa 1 (RP1) gene and the rhodopsin (RHO) gene in Chinese patients with retinitis pigmentosa sine pigmento (RPSP) and describe the genotype-phenotype relationship of the mutations. Twenty affected, unrelated Chinese individuals with RPSP (4 autosomal dominant RPSP, 12 autosomal recessive RPSP and 4 unknown inheritance pattern) were recruited between 2009 and 2012. The clinical features were determined by complete ophthalmologic examinations. Polymerase chain reaction (PCR) and direct DNA sequencing were used to screen the entire coding region and splice junctions of the RP1 gene and the RHO gene. The cosegregation analysis and population frequency studies were performed for patients with identified mutations. Five variants in the RP1 gene and one in the RHO gene were detected in 20 probands. Four missense changes (rs444772, rs446227, rs414352, rs441800) and one non-coding variant (rs56340615) were common SNPs and none of them showed a significant relationship with RPSP. A missense mutation p.R1443W was identified in the RP1 gene in three affected individuals from a family with autosomal dominant RPSP and was found to cosegregate with the phenotype in this family, suggestive of pathogenic. In addition, population frequency analysis showed the p.R1443W mutation was absent in 300 healthy controls. The identification of p.R1443W mutation cosegregating in a family with autosomal dominant RPSP highlights an atypical phenotype of the RP1 gene mutation, while RHO gene is not associated with the pathogenesis of RPSP in this study. To our knowledge, this is the fist mutation identified to associate with RPSP.

  1. Identification of a novel LMF1 nonsense mutation responsible for severe hypertriglyceridemia by targeted next-generation sequencing.

    Science.gov (United States)

    Cefalù, Angelo B; Spina, Rossella; Noto, Davide; Ingrassia, Valeria; Valenti, Vincenza; Giammanco, Antonina; Fayer, Francesca; Misiano, Gabriella; Cocorullo, Gianfranco; Scrimali, Chiara; Palesano, Ornella; Altieri, Grazia I; Ganci, Antonina; Barbagallo, Carlo M; Averna, Maurizio R

    Severe hypertriglyceridemia (HTG) may result from mutations in genes affecting the intravascular lipolysis of triglyceride (TG)-rich lipoproteins. The aim of this study was to develop a targeted next-generation sequencing panel for the molecular diagnosis of disorders characterized by severe HTG. We developed a targeted customized panel for next-generation sequencing Ion Torrent Personal Genome Machine to capture the coding exons and intron/exon boundaries of 18 genes affecting the main pathways of TG synthesis and metabolism. We sequenced 11 samples of patients with severe HTG (TG>885 mg/dL-10 mmol/L): 4 positive controls in whom pathogenic mutations had previously been identified by Sanger sequencing and 7 patients in whom the molecular defect was still unknown. The customized panel was accurate, and it allowed to confirm genetic variants previously identified in all positive controls with primary severe HTG. Only 1 patient of 7 with HTG was found to be carrier of a homozygous pathogenic mutation of the third novel mutation of LMF1 gene (c.1380C>G-p.Y460X). The clinical and molecular familial cascade screening allowed the identification of 2 additional affected siblings and 7 heterozygous carriers of the mutation. We showed that our targeted resequencing approach for genetic diagnosis of severe HTG appears to be accurate, less time consuming, and more economical compared with traditional Sanger resequencing. The identification of pathogenic mutations in candidate genes remains challenging and clinical resequencing should mainly intended for patients with strong clinical criteria for monogenic severe HTG. Copyright © 2017 National Lipid Association. Published by Elsevier Inc. All rights reserved.

  2. Prioritized Identification of Attractive and Romantic Partner Faces in Rapid Serial Visual Presentation.

    Science.gov (United States)

    Nakamura, Koyo; Arai, Shihoko; Kawabata, Hideaki

    2017-11-01

    People are sensitive to facial attractiveness because it is an important biological and social signal. As such, our perceptual and attentional system seems biased toward attractive faces. We tested whether attractive faces capture attention and enhance memory access in an involuntary manner using a dual-task rapid serial visual presentation (dtRSVP) paradigm, wherein multiple faces were successively presented for 120 ms. In Experiment 1, participants (N = 26) were required to identify two female faces embedded in a stream of animal faces as distractors. The results revealed that identification of the second female target (T2) was better when it was attractive compared to neutral or unattractive. In Experiment 2, we investigated whether perceived attractiveness affects T2 identification (N = 27). To this end, we performed another dtRSVP task involving participants in a romantic partnership with the opposite sex, wherein T2 was their romantic partner's face. The results demonstrated that a romantic partner's face was correctly identified more often than was the face of a friend or unknown person. Furthermore, the greater the intensity of passionate love participants felt for their partner (as measured by the Passionate Love Scale), the more often they correctly identified their partner's face. Our experiments indicate that attractive and romantic partners' faces facilitate the identification of the faces in an involuntary manner.

  3. Rapid Electrochemical Detection and Identification of Microbiological and Chemical Contaminants for Manned Spaceflight Project

    Science.gov (United States)

    Pierson, Duane; Botkin, Douglas; Gazda, Daniel

    2014-01-01

    Microbial control in the spacecraft environment is a daunting task, especially in the presence of human crew members. Currently, assessing the potential crew health risk associated with a microbial contamination event requires return of representative environmental samples that are analyzed in a ground-based laboratory. It is therefore not currently possible to quickly identify microbes during spaceflight. This project addresses the unmet need for spaceflight-compatible microbial identification technology. The electrochemical detection and identification platform is expected to provide a sensitive, specific, and rapid sample-to-answer capability for in-flight microbial monitoring that can distinguish between related microorganisms (pathogens and non-pathogens) as well as chemical contaminants. This will dramatically enhance our ability to monitor the spacecraft environment and the health risk to the crew. Further, the project is expected to eliminate the need for sample return while significantly reducing crew time required for detection of multiple targets. Initial work will focus on the optimization of bacterial detection and identification. The platform is designed to release nucleic acids (DNA and RNA) from microorganisms without the use of harmful chemicals. Bacterial DNA or RNA is captured by bacteria-specific probe molecules that are bound to a microelectrode, and that capture event can generate a small change in the electrical current (Lam, et al. 2012. Anal. Chem. 84(1): 21-5.). This current is measured, and a determination is made whether a given microbe is present in the sample analyzed. Chemical detection can be accomplished by directly applying a sample to the microelectrode and measuring the resulting current change. This rapid microbial and chemical detection device is designed to be a low-cost, low-power platform anticipated to be operated independently of an external power source, characteristics optimal for manned spaceflight and areas where power

  4. Rapid identification of ST131 Escherichia coli by a novel multiplex real-time allelic discrimination assay.

    Science.gov (United States)

    François, Patrice; Bonetti, Eve-Julie; Fankhauser, Carolina; Baud, Damien; Cherkaoui, Abdessalam; Schrenzel, Jacques; Harbarth, Stephan

    2017-09-01

    Escherichia coli sequence type 131 is increasingly described in severe hospital infections. We developed a rapid real-time allelic discrimination assay for the rapid identification of E. coli ST131 isolates. This rapid assay represents an affordable alternative to sequence-based strategies before completing characterization of potentially highly virulent isolates of E. coli. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. Rapid Detection and Identification of Human Hookworm Infections through High Resolution Melting (HRM) Analysis

    Science.gov (United States)

    Ngui, Romano; Lim, Yvonne A. L.; Chua, Kek Heng

    2012-01-01

    Background Hookworm infections are still endemic in low and middle income tropical countries with greater impact on the socioeconomic and public health of the bottom billion of the world's poorest people. In this study, a real-time polymerase chain reaction (PCR) coupled with high resolution melting-curve (HRM) analysis was evaluated for an accurate, rapid and sensitive tool for species identification focusing on the five human hookworm species. Methods Real-time PCR coupled with HRM analysis targeting the second internal transcribed spacer (ITS-2) of nuclear ribosomal DNA as the genetic marker was used to identify and distinguish hookworm species in human samples. Unique and distinct characteristics of HRM patterns were produced for each of the five hookworm species. The melting curves were characterized by peaks of 79.24±0.05°C and 83.00±0.04°C for Necator americanus, 79.12±0.10°C for Ancylostoma duodenale, 79.40±0.10°C for Ancylostoma ceylanicum, 79.63±0.05°C for Ancylostoma caninum and 79.70±0.14°C for Ancylostoma braziliense. An evaluation of the method's sensitivity and specificity revealed that this assay was able to detect as low as 0.01 ng/µl hookworm DNA and amplification was only recorded for hookworm positive samples. Conclusion The HRM assay developed in this study is a rapid and straightforward method for the diagnosis, identification and discrimination of five human hookworms. This assay is simple compared to other probe-based genotyping methods as it does not require multiplexing, DNA sequencing or post-PCR processing. Therefore, this method offers a new alternative for rapid detection of human hookworm species. PMID:22844538

  6. Rapid identification of closely related muscle foods by vibrational spectroscopy and machine learning.

    Science.gov (United States)

    Ellis, David I; Broadhurst, David; Clarke, Sarah J; Goodacre, Royston

    2005-12-01

    Muscle foods are an integral part of the human diet and during the last few decades consumption of poultry products in particular has increased significantly. It is important for consumers, retailers and food regulatory bodies that these products are of a consistently high quality, authentic, and have not been subjected to adulteration by any lower-grade material either by accident or for economic gain. A variety of methods have been developed for the identification and authentication of muscle foods. However, none of these are rapid or non-invasive, all are time-consuming and difficulties have been encountered in discriminating between the commercially important avian species. Whilst previous attempts have been made to discriminate between muscle foods using infrared spectroscopy, these have had limited success, in particular regarding the closely related poultry species, chicken and turkey. Moreover, this study includes novel data since no attempts have been made to discriminate between both the species and the distinct muscle groups within these species, and this is the first application of Raman spectroscopy to the study of muscle foods. Samples of pre-packed meat and poultry were acquired and FT-IR and Raman measurements taken directly from the meat surface. Qualitative interpretation of FT-IR and Raman spectra at the species and muscle group levels were possible using discriminant function analysis. Genetic algorithms were used to elucidate meaningful interpretation of FT-IR results in (bio)chemical terms and we show that specific wavenumbers, and therefore chemical species, were discriminatory for each type (species and muscle) of poultry sample. We believe that this approach would aid food regulatory bodies in the rapid identification of meat and poultry products and shows particular potential for rapid assessment of food adulteration.

  7. Rapid detection and identification of human hookworm infections through high resolution melting (HRM analysis.

    Directory of Open Access Journals (Sweden)

    Romano Ngui

    Full Text Available BACKGROUND: Hookworm infections are still endemic in low and middle income tropical countries with greater impact on the socioeconomic and public health of the bottom billion of the world's poorest people. In this study, a real-time polymerase chain reaction (PCR coupled with high resolution melting-curve (HRM analysis was evaluated for an accurate, rapid and sensitive tool for species identification focusing on the five human hookworm species. METHODS: Real-time PCR coupled with HRM analysis targeting the second internal transcribed spacer (ITS-2 of nuclear ribosomal DNA as the genetic marker was used to identify and distinguish hookworm species in human samples. Unique and distinct characteristics of HRM patterns were produced for each of the five hookworm species. The melting curves were characterized by peaks of 79.24±0.05°C and 83.00±0.04°C for Necator americanus, 79.12±0.10°C for Ancylostoma duodenale, 79.40±0.10°C for Ancylostoma ceylanicum, 79.63±0.05°C for Ancylostoma caninum and 79.70±0.14°C for Ancylostoma braziliense. An evaluation of the method's sensitivity and specificity revealed that this assay was able to detect as low as 0.01 ng/µl hookworm DNA and amplification was only recorded for hookworm positive samples. CONCLUSION: The HRM assay developed in this study is a rapid and straightforward method for the diagnosis, identification and discrimination of five human hookworms. This assay is simple compared to other probe-based genotyping methods as it does not require multiplexing, DNA sequencing or post-PCR processing. Therefore, this method offers a new alternative for rapid detection of human hookworm species.

  8. Rapid identification of emerging human-pathogenic Sporothrix species with rolling circle amplification

    Directory of Open Access Journals (Sweden)

    Anderson Messias Rodrigues

    2015-12-01

    Full Text Available Sporothrix infections are emerging as an important human and animal threat among otherwise healthy patients, especially in Brazil and China. Correct identification of sporotrichosis agents is beneficial for epidemiological surveillance, enabling implementation of adequate public-health policies and guiding antifungal therapy. In areas of limited resources where sporotrichosis is endemic, high-throughput detection methods that are specific and sensitive are preferred over phenotypic methods that usually result in misidentification of closely related Sporothrix species. We sought to establish rolling circle amplification (RCA as a low-cost screening tool for species-specific identification of human-pathogenic Sporothrix. We developed six species-specific padlock probes targeting polymorphisms in the gene encoding calmodulin. BLAST-searches revealed candidate probes that were conserved intraspecifically; no significant homology with sequences from humans, mice, plants or microorganisms outside members of Sporothrix were found. The accuracy of our RCA-based assay was demonstrated through the specificity of probe-template binding to 25 S. brasiliensis, 58 S. schenckii, 5 S. globosa, 1 S. luriei, 4 S. mexicana, and 3 S. pallida samples. No cross reactivity between closely related species was evident in vitro, and padlock probes yielded 100% specificity and sensitivity down to 3 x 10 6 copies of the target sequence. RCA-based speciation matched identifications via phylogenetic analysis of the gene encoding calmodulin and the rDNA operon (kappa 1.0; 95% confidence interval 1.0-1.0, supporting its use as a reliable alternative to DNA sequencing. This method is a powerful tool for rapid identification and specific detection of medically relevant Sporothrix, and due to its robustness has potential for ecological studies.

  9. Rapid

    Directory of Open Access Journals (Sweden)

    Nahla M. Wassim

    2013-01-01

    Full Text Available Members of Aedes caspius mosquitoes are incriminated to be a potential reservoir of “Rift Valley Fever Virus” (RVF during interepizootic periods in Egypt. Ae. caspius contains two distinct forms which are morphologically indistinguishable but differ in physiology and behavior; Ae. caspius form (a requires a blood meal for each egg batch(anautogeny, is unable to mate in confined spaces(eurygamous. The second form (b lays egg batch without blood meal (autogenous and can mate in confined spaces (stenogamous. In this work, we collected the autogenous and anautogenous forms of Ae. caspius from two different breeding habitats in the Qalyubia Governorate. Analysis of the Drosophila ace-Orthologous acetylecholinesterase gene revealed that a single polymorphic region characterized each species. Based on this region, specific primers were used to amplify the entire section of intron II, sections of Exon 2 and Exon 3 of ace-2 gene for differentiating the complex species of mosquitoes. The amplicons of anautogenous form sized 441 pb and increase 116 bp than autogenous form of Ae. caspius. High rates of point mutations were addressed; deletion/insertion events are 120 bases. The transversion mutations were 44 bases and were relatively close to the transtion mutations 43 base. The genetic distance was 0.01 between the two forms.

  10. PSP: rapid identification of orthologous coding genes under positive selection across multiple closely related prokaryotic genomes.

    Science.gov (United States)

    Su, Fei; Ou, Hong-Yu; Tao, Fei; Tang, Hongzhi; Xu, Ping

    2013-12-27

    With genomic sequences of many closely related bacterial strains made available by deep sequencing, it is now possible to investigate trends in prokaryotic microevolution. Positive selection is a sub-process of microevolution, in which a particular mutation is favored, causing the allele frequency to continuously shift in one direction. Wide scanning of prokaryotic genomes has shown that positive selection at the molecular level is much more frequent than expected. Genes with significant positive selection may play key roles in bacterial adaption to different environmental pressures. However, selection pressure analyses are computationally intensive and awkward to configure. Here we describe an open access web server, which is designated as PSP (Positive Selection analysis for Prokaryotic genomes) for performing evolutionary analysis on orthologous coding genes, specially designed for rapid comparison of dozens of closely related prokaryotic genomes. Remarkably, PSP facilitates functional exploration at the multiple levels by assignments and enrichments of KO, GO or COG terms. To illustrate this user-friendly tool, we analyzed Escherichia coli and Bacillus cereus genomes and found that several genes, which play key roles in human infection and antibiotic resistance, show significant evidence of positive selection. PSP is freely available to all users without any login requirement at: http://db-mml.sjtu.edu.cn/PSP/. PSP ultimately allows researchers to do genome-scale analysis for evolutionary selection across multiple prokaryotic genomes rapidly and easily, and identify the genes undergoing positive selection, which may play key roles in the interactions of host-pathogen and/or environmental adaptation.

  11. Age-Related Declines in Early Sensory Memory: Identification of Rapid Auditory and Visual Stimulus Sequences.

    Science.gov (United States)

    Fogerty, Daniel; Humes, Larry E; Busey, Thomas A

    2016-01-01

    Age-related temporal-processing declines of rapidly presented sequences may involve contributions of sensory memory. This study investigated recall for rapidly presented auditory (vowel) and visual (letter) sequences presented at six different stimulus onset asynchronies (SOA) that spanned threshold SOAs for sequence identification. Younger, middle-aged, and older adults participated in all tasks. Results were investigated at both equivalent performance levels (i.e., SOA threshold) and at identical physical stimulus values (i.e., SOAs). For four-item sequences, results demonstrated best performance for the first and last items in the auditory sequences, but only the first item for visual sequences. For two-item sequences, adults identified the second vowel or letter significantly better than the first. Overall, when temporal-order performance was equated for each individual by testing at SOA thresholds, recall accuracy for each position across the age groups was highly similar. These results suggest that modality-specific processing declines of older adults primarily determine temporal-order performance for rapid sequences. However, there is some evidence for a second amodal processing decline in older adults related to early sensory memory for final items in a sequence. This selective deficit was observed particularly for longer sequence lengths and was not accounted for by temporal masking.

  12. Rapid identification of HPV 16 and 18 by multiplex nested PCR-immunochromatographic test.

    Science.gov (United States)

    Kuo, Yung-Bin; Li, Yi-Shuan; Chan, Err-Cheng

    2015-02-01

    Human papillomavirus (HPV) types 16 and 18 are known to be high-risk viruses that cause cervical cancer. An HPV rapid testing kit that could help physicians to make early and more informed decisions regarding patient care is needed urgently but not yet available. This study aimed to develop a multiplex nested polymerase chain reaction-immunochromatographic test (PCR-ICT) for the rapid identification of HPV 16 and 18. A multiplex nested PCR was constructed to amplify the HPV 16 and 18 genotype-specific L1 gene fragments and followed by ICT which coated with antibodies to identify rapidly the different PCR products. The type-specific gene regions of high-risk HPV 16 and 18 could be amplified successfully by multiplex nested PCR at molecular sizes of approximately 99 and 101bp, respectively. The capture antibodies raised specifically against the moleculars labeled on the PCR products could be detected simultaneously both HPV 16 and 18 in one strip. Under optimal conditions, this PCR-ICT assay had the capability to detect HPV in a sample with as low as 100 copies of HPV viral DNA. The PCR-ICT system has the advantage of direct and simultaneous detection of two high-risk HPV 16 and 18 DNA targets in one sample, which suggested a significant potential of this assay for clinical application. Copyright © 2014. Published by Elsevier B.V.

  13. Rapid detection of ERG11 gene mutations in clinical Candida albicans isolates with reduced susceptibility to fluconazole by rolling circle amplification and DNA sequencing

    OpenAIRE

    Wang, Huiping; Kong, Fanrong; Sorrell, Tania C; Wang, Bin; McNicholas, Paul; Pantarat, Namfon; Ellis, David; Xiao, Meng; Widmer, Fred; Chen, Sharon CA

    2009-01-01

    Abstract Background Amino acid substitutions in the target enzyme Erg11p of azole antifungals contribute to clinically-relevant azole resistance in Candida albicans. A simple molecular method for rapid detection of ERG11 gene mutations would be an advantage as a screening tool to identify potentially-resistant strains and to track their movement. To complement DNA sequencing, we developed a padlock probe and rolling circle amplification (RCA)-based method to detect a series of mutations in th...

  14. Identification and verification of a pathogenic MLH1 mutation c.1145dupA in a Lynch syndrome family

    Directory of Open Access Journals (Sweden)

    Huang Feifei

    2017-06-01

    Full Text Available Lynch syndrome (LS, an autosomal-dominant disorder with an increased risk of predominantly colorectal and endometrial cancers, is caused by germ-line mutations in mismatch repair genes. The identification of germ-line mutations that predispose to cancer is important to further our understanding of tumorigenesis, guide patient management and inform the best practice for healthcare. A 45-year-old woman with atypical endometrial hyperplasia who suffered colon cancer at the age of 30 years underwent hysterectomy and genetic counseling. Pedigree analysis revealed her family fulfilling the Amsterdam I criteria. Next-generation sequencing was offered to the patient. A mutation in the MLH1 gene, c.1145dupA, was identified and verified by Sanger sequencing. In addition, her nine family members were tested for the mutation. Two were affected (colon cancer at the age of 43 years and 45 years and one healthy relative carried the same mutation in the MLH1 gene. The mutation resulted in a frame-shift (p.Met383Aspfs*12 located in exon12, as well as a polypeptide truncation of 393 amino acids by the formation of a premature stop codon. An immunohistochemistry analysis of endometrial hyperplasia tissues revealed defects in MLH1 and PMS2 protein expression in the patient. Based on the 2015 American College of Medical Genetics and Genomics (ACMG guideline, we report this MLH1 c.1145dupA variation to be a pathogenic mutation that contributes to a strongly increased cancer risk in this LS family. Proper screening suggestions were offered to the three affected patients and the healthy carrier. To the best of our knowledge, this germ-line mutation of MLH1 was previously submitted to the Leiden Open Variation Database (LOVD database, but no comprehensive evidence or supporting observations were reported previously in the literature. The present report found a single nucleotide insertion in exon12 of the MLH1 gene, which can be considered causative of Lynch phenotype

  15. Identification of Mutations Underlying 20 Inborn Errors of Metabolism in the United Arab Emirates Population

    Science.gov (United States)

    Ben-Rebeh, Imen; Hertecant, Jozef L.; Al-Jasmi, Fatma A.; Aburawi, Hanan E.; Al-Yahyaee, Said A.; Al-Gazali, Lihadh

    2012-01-01

    Inborn errors of metabolism (IEM) are frequently encountered by physicians in the United Arab Emirates (UAE). However, the mutations underlying a large number of these disorders have not yet been determined. Therefore, the objective of this study was to identify the mutations underlying a number of IEM disorders among UAE residents from both national and expatriate families. A case series of patients from 34 families attending the metabolic clinic at Tawam Hospital were clinically evaluated, and molecular testing was carried out to determine their causative mutations. The mutation analysis was carried out at molecular genetics diagnostic laboratories. Thirty-eight mutations have been identified as responsible for twenty IEM disorders, including in the metabolism of amino acids, lipids, steroids, metal transport and mitochondrial energy metabolism, and lysosomal storage disorders. Nine of the identified mutations are novel, including two missense mutations, three premature stop codons and four splice site mutations. Mutation analysis of IEM disorders in the UAE population has an important impact on molecular diagnosis and genetic counseling for families affected by these disorders. PMID:22106832

  16. Identification of four novel mutations of the WFS1 gene in Iranian Wolfram syndrome pedigrees.

    Science.gov (United States)

    Ghahraman, Martha; Abbaszadegan, Mohammad Reza; Vakili, Rahim; Hosseini, Sousan; Fardi Golyan, Fatemeh; Ghaemi, Nosrat; Forghanifard, Mohammad Mahdi

    2016-12-01

    Wolfram syndrome is a rare neurodegenerative disorder with an autosomal recessive pattern of inheritance characterized by various clinical manifestations. The related gene, WFS1, encodes a transmembrane glycoprotein, named wolframin. Genetic analyses demonstrated that mutations in this gene are associated with WS type 1. Our aim in this study was to sequence WFS1 coding region in Iranian Wolfram syndrome pedigrees. Genomic DNA was extracted from peripheral blood of 12 WS patients and their healthy parents. Exons 2-8 and the exon-intron junctions of WFS1 were sequenced. DNA sequences were compared to the reference using Sequencher software. Molecular analysis of WFS1 revealed six different mutations. Four novel and two previously reported mutations were identified. One novel mutation, c.1379_1381del, is predicted to produce an aberrant protein. A second novel mutation, c.1384G > T, encodes a truncated protein. Novel mutation, c.1097-1107dup (11 bp), causes a frameshift which results in a premature stop codon. We screened for the novel missense mutation, c.1010C > T, in 100 control alleles. This mutation was not found in any of the healthy controls. Our study increased the spectrum of WFS1 mutations and supported the role of WFS1 in susceptibility to WS. We hope that these findings open new horizons to future molecular investigations which may help to prevent and treat this devastating disease.

  17. Identification of new biomarker of radiation exposure for establishing rapid, simplified biodosimetric method

    International Nuclear Information System (INIS)

    Iizuka, Daisuke; Kawai, Hidehiko; Kamiya, Kenji; Suzuki, Fumio; Izumi, Shunsuke

    2014-01-01

    Until now, counting chromosome aberration is the most accurate method for evaluating radiation doses. However, this method is time consuming and requires skills for evaluating chromosome aberrations. It could be difficult to apply this method to majority of people who are expected to be exposed to ionizing radiation. In this viewpoint, establishment of rapid, simplified biodosimetric methods for triage will be anticipated. Due to the development of mass spectrometry method and the identification of new molecules such as microRNA (miRNA), it is conceivable that new molecular biomarker of radiation exposure using some newly developed mass spectrometry. In this review article, the part of our results including the changes of protein (including the changes of glycosylation), peptide, metabolite, miRNA after radiation exposure will be shown. (author)

  18. A PCR detection method for rapid identification of Melissococcus pluton in honeybee larvae.

    Science.gov (United States)

    Govan, V A; Brözel, V; Allsopp, M H; Davison, S

    1998-05-01

    Melissococcus pluton is the causative agent of European foulbrood, a disease of honeybee larvae. This bacterium is particularly difficult to isolate because of its stringent growth requirements and competition from other bacteria. PCR was used selectively to amplify specific rRNA gene sequences of M. pluton from pure culture, from crude cell lysates, and directly from infected bee larvae. The PCR primers were designed from M. pluton 16S rRNA sequence data. The PCR products were visualized by agarose gel electrophoresis and confirmed as originating from M. pluton by sequencing in both directions. Detection was highly specific, and the probes did not hybridize with DNA from other bacterial species tested. This method enabled the rapid and specific detection and identification of M. pluton from pure cultures and infected bee larvae.

  19. Rapid identification of pathogenic streptococci isolated from moribund red tilapia (Oreochromis spp.).

    Science.gov (United States)

    Abdelsalam, Mohamed; Elgendy, Mamdouh Y; Shaalan, Mohamed; Moustafa, Mohamed; Fujino, Masayuki

    2017-03-01

    Accurate and rapid identification of bacterial pathogens of fish is essential for the effective treatment and speedy control of infections. Massive mortalities in market-sized red tilapia (Oreochromis spp.) were noticed in mariculture concrete ponds in northern Egypt. Histopathological examination revealed marked congestion in the central vein of the liver with the presence of bacterial aggregates inside the lumen and in the vicinity of the central vein. A total of 12 isolates of streptococci were obtained from the moribund fish. This study documented the ability of the MicroSeq 500 16S bacterial sequencing method to accurately identify Streptococcus agalactiae and S. dysgalactiae mixed infections from moribund red tilapia that were difficult to be recognised by the commercial biochemical systems. The continuously decreasing cost of the sequencing technique should encourage its application in routine diagnostic procedures.

  20. The identification of point mutations in Duchenne muscular dystrophy patients by using reverse-transcription PCR and the protein truncation test

    Energy Technology Data Exchange (ETDEWEB)

    Gardner, R.J.; Bobrow, M.; Roberts, R.G. [St. Thomas`s Hospitals, London (United Kingdom)

    1995-08-01

    The protein truncation test (PTT) is a mutation-detection method that monitors the integrity of the open reading frame (ORF). More than 60% of cases of Duchenne muscular dystrophy (DMD) result from gross frameshifting deletions in the dystrophin gene that are detectable by multiplex PCR system. It has become apparent that virtually all of the remaining DMD mutations also disrupt the translational reading frame, making the PTT a logical next step toward a comprehensive strategy for the identification of all DMD mutations. We report here a pilot study involving 22 patients and describe the mutations characterized. These constitute 12 point mutations or small insertions/deletions and 4 gross rearrangements. We also have a remaining five patients in whom there does not appear to be mutation in the ORF. We believe that reverse-transcription-PCR/PTT is an efficient method by which to screen for small mutations in DMD patients with no deletion. 29 refs., 2 figs., 3 tabs.

  1. Rapid direct identification of Cryptococcus neoformans from pigeon droppings by nested PCR using CNLAC1 gene.

    Science.gov (United States)

    Chae, H S; Park, G N; Kim, S H; Jo, H J; Kim, J T; Jeoung, H Y; An, D J; Kim, N H; Shin, B W; Kang, Y I; Chang, K S

    2012-08-01

    Isolation and identification of Cryptococcus neoformans and pathogenic yeast-like fungi from pigeon droppings has been taken for a long time and requires various nutrients for its growth. In this study, we attempted to establish a rapid direct identification method of Cr. neoformans from pigeon dropping samples by nested-PCR using internal transcribed spacer (ITS) CAP64 and CNLAC1 genes, polysaccharide capsule gene and laccase-associated gene to produce melanin pigment, respectively, which are common genes of yeasts. The ITS and CAP64 genes were amplified in all pathogenic yeasts, but CNLAC1 was amplified only in Cr. neoformans. The ITS gene was useful for yeast genotyping depending on nucleotide sequence. Homology of CAP64 genes among the yeasts were very high. The specificity of PCR using CNLAC1 was demonstrated in Cr. neoformans environmental strains but not in other yeast-like fungi. The CNLAC1 gene was detected in 5 serotypes of Cr. neoformans. The nested-PCR amplified up to 10(-11) μg of the genomic DNA and showed high sensitivity. All pigeon droppings among 31 Cr. neoformans-positive samples were positive and all pigeon droppings among 348 Cr. neoformans-negative samples were negative by the direct nested-PCR. In addition, after primary enrichment of pigeon droppings in Sabouraud dextrose broth, all Cr. neoformans-negative samples were negative by the nested-PCR, which showed high specificity. The nested-PCR showed high sensitivity without culture of pigeon droppings. Nested-PCR using CNLAC1 provides a rapid and reliable molecular diagnostic method to overcome weak points such as long culture time of many conventional methods.

  2. Rapid and Simple Detection of Hot Spot Point Mutations of Epidermal Growth Factor Receptor, BRAF, and NRAS in Cancers Using the Loop-Hybrid Mobility Shift Assay

    Science.gov (United States)

    Matsukuma, Shoichi; Yoshihara, Mitsuyo; Kasai, Fumio; Kato, Akinori; Yoshida, Akira; Akaike, Makoto; Kobayashi, Osamu; Nakayama, Haruhiko; Sakuma, Yuji; Yoshida, Tsutomu; Kameda, Yoichi; Tsuchiya, Eiju; Miyagi, Yohei

    2006-01-01

    A simple and rapid method to detect the epidermal growth factor receptor hot spot mutation L858R in lung adenocarcinoma was developed based on principles similar to the universal heteroduplex generator technology. A single-stranded oligonucleotide with an internal deletion was used to generate heteroduplexes (loop-hybrids) bearing a loop in the complementary strand derived from the polymerase chain reaction product of the normal or mutant allele. By placing deletion in the oligonucleotide adjacent to the mutational site, difference in electrophoretic mobility between loop-hybrids with normal and mutated DNA was distinguishable in a native polyacrylamide gel. The method was also modified to detect in-frame deletion mutations of epidermal growth factor receptor in lung adenocarcinomas. In addition, the method was adapted to detect hot spot mutations in the B-type Raf kinase (BRAF) at V600 and in a Ras-oncogene (NRAS) at Q61, the mutations commonly found in thyroid carcinomas. Our mutation detection system, designated the loop-hybrid mobility shift assay was sensitive enough to detect mutant DNA comprising 7.5% of the total DNA. As a simple and straightforward mutation detection technique, loop-hybrid mobility shift assay may be useful for the molecular diagnosis of certain types of clinical cancers. Other applications are also discussed. PMID:16931592

  3. Rapid screening of ASXL1, IDH1, IDH2, and c-CBL mutations in de novo acute myeloid leukemia by high-resolution melting.

    Science.gov (United States)

    Ibáñez, Mariam; Such, Esperanza; Cervera, José; Luna, Irene; Gómez-Seguí, Inés; López-Pavía, María; Dolz, Sandra; Barragán, Eva; Fuster, Oscar; Llop, Marta; Rodríguez-Veiga, Rebeca; Avaria, Amparo; Oltra, Silvestre; Senent, M Leonor; Moscardó, Federico; Montesinos, Pau; Martínez-Cuadrón, David; Martín, Guillermo; Sanz, Miguel A

    2012-11-01

    Recently, many novel molecular abnormalities were found to be distinctly associated with acute myeloid leukemia (AML). However, their clinical relevance and prognostic implications are not well established. We developed a new combination of high-resolution melting assays on a LightCycler 480 and direct sequencing to detect somatic mutations of ASXL1 (exon 12), IDH1 (exon 4), IDH2 (exon 4), and c-CBL (exons 8 and 9) genes to know their incidence and prognostic effect in a cohort of 175 patients with de novo AML: 16 patients (9%) carried ASXL1 mutations, 16 patients had IDH variations (3% with IDH1(R132) and 6% with IDH2(R140)), and none had c-CBL mutations. Patients with ASXL1 mutations did not harbor IDH1, [corrected] or CEBPA mutations, and a combination of ASXL1 and IDH2 mutations was found only in one patient. In addition, we did not find IDH1 and FLT3 or CEBPA mutations concurrently or IDH2 with CEBPA. IDH1 and IDH2 mutations were mutually exclusive. Alternatively, NPM1 mutations were concurrently found with ASXL1, IDH1, or IDH2 with a variable incidence. Mutations were not significantly correlated with any of the clinical and biological features studied. High-resolution melting is a reliable, rapid, and efficient screening technique for mutation detection in AML. The incidence for the studied genes was in the range of those previously reported. We were unable to find an effect on the outcome. Copyright © 2012 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.

  4. Rapid and Direct VHH and Target Identification by Staphylococcal Surface Display Libraries

    Directory of Open Access Journals (Sweden)

    Marco Cavallari

    2017-07-01

    Full Text Available Unbiased and simultaneous identification of a specific antibody and its target antigen has been difficult without prior knowledge of at least one interaction partner. Immunization with complex mixtures of antigens such as whole organisms and tissue extracts including tumoral ones evokes a highly diverse immune response. During such a response, antibodies are generated against a variety of epitopes in the mixture. Here, we propose a surface display design that is suited to simultaneously identify camelid single domain antibodies and their targets. Immune libraries of single-domain antigen recognition fragments from camelid heavy chain-only antibodies (VHH were attached to the peptidoglycan of Gram-positive Staphylococcus aureus employing its endogenous housekeeping sortase enzyme. The sortase transpeptidation reaction covalently attached the VHH to the bacterial peptidoglycan. The reversible nature of the reaction allowed the recovery of the VHH from the bacterial surface and the use of the VHH in downstream applications. These staphylococcal surface display libraries were used to rapidly identify VHH as well as their targets by immunoprecipitation (IP. Our novel bacterial surface display platform was stable under harsh screening conditions, allowed fast target identification, and readily permitted the recovery of the displayed VHH for downstream analysis.

  5. Rapid experimental SAD phasing and hot-spot identification with halogenated fragments

    Energy Technology Data Exchange (ETDEWEB)

    Bauman, Joseph D.; Harrison, Jerry Joe E. K.; Arnold, Eddy

    2016-01-01

    Through X-ray crystallographic fragment screening, 4-bromopyrazole was discovered to be a `magic bullet' that is capable of binding at many of the ligand `hot spots' found in HIV-1 reverse transcriptase (RT). The binding locations can be in pockets that are `hidden' in the unliganded crystal form, allowing rapid identification of these sites forin silicoscreening. In addition to hot-spot identification, this ubiquitous yet specific binding provides an avenue for X-ray crystallographic phase determination, which can be a significant bottleneck in the determination of the structures of novel proteins. The anomalous signal from 4-bromopyrazole or 4-iodopyrazole was sufficient to determine the structures of three proteins (HIV-1 RT, influenza A endonuclease and proteinase K) by single-wavelength anomalous dispersion (SAD) from single crystals. Both compounds are inexpensive, readily available, safe and very soluble in DMSO or water, allowing efficient soaking into crystals.

  6. Performance of chromogenic media for Candida in rapid presumptive identification of Candida species from clinical materials.

    Science.gov (United States)

    Pravin Charles, M V; Kali, Arunava; Joseph, Noyal Mariya

    2015-06-01

    In perspective of the worldwide increase in a number of immunocompromised patients, the need for identification of Candida species has become a major concern. The development of chromogenic differential media, introduced recently, facilitate rapid speciation. However, it can be employed for routine mycology workup only after an exhaustive evaluation of its benefit and cost effectiveness. This study was undertaken to evaluate the benefit and cost effectiveness of chromogenic media for speciation of Candida clinical isolates. Sputum samples of 382 patients were screened for the presence of Candida spp. by Gram stain and culture on sabouraud dextrose agar. Candida species were identified using Gram stain morphology, germ tube formation, cornmeal agar with Tween-80, sugar fermentation tests and morphology on HiCrome Candida differential agar. All the Candida isolates were inoculated on HiCrome Candida agar (HiMedia, Mumbai, India). The sensitivity and specificity of HiCrome agar for identification of Candida albicans were 90% and 96.42%, respectively whereas sensitivity and specificity of carbohydrate fermentation test were 86.67% and 74.07%, respectively. Sensitivity and specificity values of HiCrome agar for detection of C. albicans, Candida parapsilosis and Candida glabrata were above 90%. We found HiCrome agar has high sensitivity and specificity comparable to that of the conventional method. In addition, use of this differential media could significantly cut down the turnaround time as well as cost of sample processing.

  7. Rapid paper disk test for identification of Helicobacter pylori in mixed cultures of gerbil gastric homogenates.

    Science.gov (United States)

    Castillo-Juarez, Israel; Rangel-Vega, Adrian; Romero, Irma

    2010-10-01

    A method denominated rapid paper disk test (RPDT) was developed to identify H. pylori colonies in complex cultures obtained from gerbil gastric homogenates. Identification is based on a characteristic reaction pattern (RP) for H. pylori colonies given by the combination of the urease-oxidase activities on a paper disk. Compared to the RPs obtained from gerbil's intestinal tract isolated bacteria, H. pylori RP is completely distinguishable, even from those of bacteria that share one or both activities as are Aerococcus urinae, Bacillus sphaericus, Bacillus brevis, Corynebacterium pseudogenitalium, and Staphylococcus simulans, as well as from those produced by collection strains Proteus vulgaris and Pseudomonas aeruginosa. This method allows the practical quantification of H. pylori colonies in highly contaminated plates. RPDT has the following advantages over other methodologies that use indicators in the medium: it employs two of the three routinely used H. pylori biochemical identification tests, the reagents do not interfere with bacterial viability, there are no restrictions in relation to the medium used, and it is a simple, fast, and low-cost method. Copyright © 2010 Elsevier B.V. All rights reserved.

  8. Rapid testing and identification of actuator using dSPACE real-time emulator

    Science.gov (United States)

    Xie, Daocheng; Wang, Zhongwei; Zeng, Qinghua

    2011-10-01

    To solve the problem of model identification of actuator in control system design of aerocraft, testing system based on dSPACE emulator is established, sending testing signal and receiving feedback voltage are realized using dSPACE interactive cards, communication between signal generating equipment and feedback voltage acquisition equipment is synchronized. This paper introduces the hardware architecture and key technologies of the simulation system. Constructing, downloading and calculating of the testing model is finished using dSPACE emulator, D/A transfer of testing signal is realized using DS2103 card, DS2002 card transfer the feedback voltage to digital value. Filtering module is added to the signal acquisition, for reduction of noise interference in the A/D channel. Precision of time and voltage is improved by setting acquisition period 1ms. The data gathered is recorded and displayed with Controldesk tools. The response of four actuators under different frequency are tested, frequency-domain analysis is done using least square method, the model of actuator is identified, simulation data fits well with real response of the actuator. The testing system created with dSPACE emulator satisfies the rapid testing and identification of actuator.

  9. Rapid identification of acetic acid bacteria using MALDI-TOF mass spectrometry fingerprinting.

    Science.gov (United States)

    Andrés-Barrao, Cristina; Benagli, Cinzia; Chappuis, Malou; Ortega Pérez, Ruben; Tonolla, Mauro; Barja, François

    2013-03-01

    Acetic acid bacteria (AAB) are widespread microorganisms characterized by their ability to transform alcohols and sugar-alcohols into their corresponding organic acids. The suitability of matrix-assisted laser desorption-time of flight mass spectrometry (MALDI-TOF MS) for the identification of cultured AAB involved in the industrial production of vinegar was evaluated on 64 reference strains from the genera Acetobacter, Gluconacetobacter and Gluconobacter. Analysis of MS spectra obtained from single colonies of these strains confirmed their basic classification based on comparative 16S rRNA gene sequence analysis. MALDI-TOF analyses of isolates from vinegar cross-checked by comparative sequence analysis of 16S rRNA gene fragments allowed AAB to be identified, and it was possible to differentiate them from mixed cultures and non-AAB. The results showed that MALDI-TOF MS analysis was a rapid and reliable method for the clustering and identification of AAB species. Copyright © 2012 Elsevier GmbH. All rights reserved.

  10. Determining Y-STR mutation rates in deep-routing genealogies: Identification of haplogroup differences.

    Science.gov (United States)

    Claerhout, Sofie; Vandenbosch, Michiel; Nivelle, Kelly; Gruyters, Leen; Peeters, Anke; Larmuseau, Maarten H D; Decorte, Ronny

    2018-05-01

    Knowledge of Y-chromosomal short tandem repeat (Y-STR) mutation rates is essential to determine the most recent common ancestor (MRCA) in familial searching or genealogy research. Up to now, locus-specific mutation rates have been extensively examined especially for commercially available forensic Y-STRs, while haplogroup specific mutation rates have not yet been investigated in detail. Through 450 patrilineally related namesakes distributed over 212 deep-rooting genealogies, the individual mutation rates of 42 Y-STR loci were determined, including 27 forensic Y-STR loci from the Yfiler ® Plus kit and 15 additional Y-STR loci (DYS388, DYS426, DYS442, DYS447, DYS454, DYS455, DYS459a/b, DYS549, DYS607, DYS643, DYS724a/b and YCAIIa/b). At least 726 mutations were observed over 148,596 meiosis and individual Y-STR mutation rates varied from 2.83 × 10 -4 to 1.86 × 10 -2 . The mutation rate was significantly correlated with the average allele size, the complexity of the repeat motif sequence and the age of the father. Significant differences in average Y-STR mutations rates were observed when haplogroup 'I & J' (4.03 × 10 -3 mutations/generation) was compared to 'R1b' (5.35 × 10 -3 mutations/generation) and to the overall mutation rate (5.03 × 10 -3 mutations/generation). A difference in allele size distribution was identified as the only cause for these haplogroup specific mutation rates. The haplogroup specific mutation rates were also present within the commercially available Y-STR kits (Yfiler ® , PowerPlex ® Y23 System and Yfiler ® Plus). This observation has consequences for applications where an average Y-STR mutation rate is used, e.g. tMRCA estimations in familial searching and genealogy research. Copyright © 2018 Elsevier B.V. All rights reserved.

  11. Identification of novel mutations in X-linked retinitis pigmentosa families and implications for diagnostic testing

    Science.gov (United States)

    Glaus, Esther; Lorenz, Birgit; Netzer, Christian; Li, Yün; Schambeck, Maria; Wittmer, Mariana; Feil, Silke; Kirschner-Schwabe, Renate; Rosenberg, Thomas; Cremers, Frans P.M.; Bergen, Arthur A.B.; Barthelmes, Daniel; Baraki, Husnia; Schmid, Fabian; Tanner, Gaby; Fleischhauer, Johannes; Orth, Ulrike; Becker, Christian; Wegscheider, Erika; Nürnberg, Gudrun; Nürnberg, Peter; Bolz, Hanno Jörn; Gal, Andreas; Berger, Wolfgang

    2008-01-01

    Purpose The goal of this study was to identify mutations in X-chromosomal genes associated with retinitis pigmentosa (RP) in patients from Germany, The Netherlands, Denmark, and Switzerland. Methods In addition to all coding exons of RP2, exons 1 through 15, 9a, ORF15, 15a and 15b of RPGR were screened for mutations. PCR products were amplified from genomic DNA extracted from blood samples and analyzed by direct sequencing. In one family with apparently dominant inheritance of RP, linkage analysis identified an interval on the X chromosome containing RPGR, and mutation screening revealed a pathogenic variant in this gene. Patients of this family were examined clinically and by X-inactivation studies. Results This study included 141 RP families with possible X-chromosomal inheritance. In total, we identified 46 families with pathogenic sequence alterations in RPGR and RP2, of which 17 mutations have not been described previously. Two of the novel mutations represent the most 3’-terminal pathogenic sequence variants in RPGR and RP2 reported to date. In exon ORF15 of RPGR, we found eight novel and 14 known mutations. All lead to a disruption of open reading frame. Of the families with suggested X-chromosomal inheritance, 35% showed mutations in ORF15. In addition, we found five novel mutations in other exons of RPGR and four in RP2. Deletions in ORF15 of RPGR were identified in three families in which female carriers showed variable manifestation of the phenotype. Furthermore, an ORF15 mutation was found in an RP patient who additionally carries a 6.4 kbp deletion downstream of the coding region of exon ORF15. We did not identify mutations in 39 sporadic male cases from Switzerland. Conclusions RPGR mutations were confirmed to be the most frequent cause of RP in families with an X-chromosomal inheritance pattern. We propose a screening strategy to provide molecular diagnostics in these families. PMID:18552978

  12. [Identification of an ideal noninvasive method to detect A3243G gene mutation in MELAS syndrome].

    Science.gov (United States)

    Ma, Yi-nan; Fang, Fang; Yang, Yan-ling; Zhang, Ying; Wang, Song-tao; Xu, Yu-feng; Pei, Pei; Yuan, Yun; Bu, Ding-fang; Qi, Yu

    2008-12-16

    To identify a better non-invasive method to detect the carrier of mitochondrial A3243G mutation, a cause of mitochondrial encephalopathy-lactic acidosis-stroke like episode (MELAS) syndrome. DNA was extracted from the peripheral blood, urine, hair follicle, and saliva of 25 MELAS syndrome patients carrying A3243G mutation and their mothers and other maternal relatives, 33 persons in number, and the muscle tissues from 5 patients obtained by biopsy. A3243G mutation was detected by PCR-RFLP method, and the A3243G mutation ratio was identified by measuring the density of each band and calculation with the software AlphaEase 5.0. A3243G mutations were detected in all tissues of the 25 MELAS patients. The A3243G mutation ratio in urine was 62% +/- 9%, significantly higher than that in the blood [(36% +/- 10%), t = -11.13, P < 0.01]. A3243G mutations were detected in at least one tissue of the 28 maternal relatives. The A3243G mutation rates in their urine samples was 33.0% (5.0% - 70.4%), significantly higher than that in their blood samples [8.0% (0 - 33.3%), z = -4.197, P < 0.01]. There was no significant difference in A3243G mutation ratio among the samples of hair follicle, saliva, and blood. The A3243G mutation ratio in urine is significantly higher than those in blood samples of the patients and their maternal relatives. A noninvasive method, A3243G mutation ratio analysis of urine is superior to that in blood.

  13. Identification of Novel Compound Mutations in PLA2G6-Associated Neurodegeneration Patient with Characteristic MRI Imaging.

    Science.gov (United States)

    Guo, Sen; Yang, Liu; Liu, Huijie; Chen, Wei; Li, Jinchen; Yu, Ping; Sun, Zhong Sheng; Chen, Xiang; Du, Jie; Cai, Tao

    2017-08-01

    Neurodegeneration with brain iron accumulation comprises a heterogeneous group of disorders characterized clinically by progressive motor dysfunction. Accurate identification of de novo and rare inherited mutations is important for determining causative genes of undiagnosed neurological diseases. In the present study, we report a unique case with cerebellar ataxia symptoms and social communication difficulties in an intermarriage family. MRI showed a marked cerebellar atrophy and the "eye-of-the-tiger"-like sign in the medial globus pallidus. Potential genetic defects were screened by whole-exome sequencing (WES) for the patient and four additional family members. A previously undescribed de novo missense mutation (c.1634A>G, p.K545R) in the exon 12 of the PLA2G6 gene was identified. A second rare variant c.1077G>A at the end of exon 7 was also identified, which was inherited from the mother, and resulted in a frame-shift mutation (c.1074_1077del.GTCG) due to an alternative splicing. In conclusion, the identification of the "eye-of-the-tiger"-like sign in the globus pallidus of the patient expands the phenotypic spectrum of PLA2G6-associated disorders and reveals its value in differential diagnosis of PLA2G6-associated disorders.

  14. [Identification of novel pathogenic gene mutations in pediatric acute myeloid leukemia by whole-exome resequencing].

    Science.gov (United States)

    Shiba, Norio

    2015-12-01

    A new class of gene mutations, identified in the pathogenesis of adult acute myeloid leukemia (AML), includes DNMT3A, IDH1/2, TET2 and EZH2. However, these mutations are rare in pediatric AML cases, indicating that pathogeneses differ between adult and pediatric forms of AML. Meanwhile, the recent development of massively parallel sequencing technologies has provided a new opportunity to discover genetic changes across entire genomes or proteincoding sequences. In order to reveal a complete registry of gene mutations, we performed whole exome resequencing of paired tumor-normal specimens from 19 pediatric AML cases using Illumina HiSeq 2000. In total, 80 somatic mutations or 4.2 mutations per sample were identified. Many of the recurrent mutations identified in this study involved previously reported targets in AML, such as FLT3, CEBPA, KIT, CBL, NRAS, WT1 and EZH2. On the other hand, several genes were newly identified in the current study, including BCORL1 and major cohesin components such as SMC3 and RAD21. Whole exome resequencing revealed a complex array of gene mutations in pediatric AML genomes. Our results indicate that a subset of pediatric AML represents a discrete entity that could be discriminated from its adult counterpart, in terms of the spectrum of gene mutations.

  15. Cancel all Hollidays for SLX4 mutations: identification of a new Fanconi anemia subtype, FANCP.

    Science.gov (United States)

    Kang, M H

    2011-07-01

    SLX4, a coordinator of structure-specific endo-nucleases, is mutated in a new Fanconi anemia subtype Stoepker et al. (2011) Nature Genetics 43:138-141. Mutations of the SLX4 gene in Fanconi anemia Kim et al. (2011) Nature Genetics 43:142-146. © 2011 John Wiley & Sons A/S.

  16. Identification of novel mutations including a double mutation in patients with inherited cardiomyopathy by a targeted sequencing approach using the Ion Torrent PGM system.

    Science.gov (United States)

    Zhao, Yue; Cao, Hong; Song, Yindi; Feng, Yue; Ding, Xiaoxue; Pang, Mingjie; Zhang, Yunmei; Zhang, Hong; Ding, Jiahuan; Xia, Xueshan

    2016-06-01

    knowledge. This patient provided us with more information regarding the genotype-phenotype correlation between mutations of MYH7 and PRKAG2. Taken together, these findings provide insight into the molecular mechanisms underlying inherited cardiomyopathy. The mutations identified in this study may be further investigated in the future in order to improve the diagnosis and treatment of patients with inherited cardiomyopathy. Furthermore, our findings indicated that sequencing using the Ion Torrent PGM system is a useful approach for the identification of pathogenic mutations associated with inherited cardiomyopathy, and it may be used for the risk evaluation of individuals with a possible susceptibility to inherited cardiomyopathy.

  17. Rapid identification and source-tracking of Listeria monocytogenes using MALDI-TOF mass spectrometry.

    Science.gov (United States)

    Jadhav, Snehal; Gulati, Vandana; Fox, Edward M; Karpe, Avinash; Beale, David J; Sevior, Danielle; Bhave, Mrinal; Palombo, Enzo A

    2015-06-02

    Listeria monocytogenes is an important foodborne pathogen responsible for the sometimes fatal disease listeriosis. Public health concerns and stringent regulations associated with the presence of this pathogen in food and food processing environments underline the need for rapid and reliable detection and subtyping techniques. In the current study, the application of matrix assisted laser desorption/ionisation-time-of-flight mass spectrometry (MALDI-TOF MS) as a single identification and source-tracking tool for a collection of L. monocytogenes isolates, obtained predominantly from dairy sources within Australia, was explored. The isolates were cultured on different growth media and analysed using MALDI-TOF MS at two incubation times (24 and 48 h). Whilst reliable genus-level identification was achieved from most media, identification at the species level was found to be dependent on culture conditions. Successful speciation was highest for isolates cultured on the chromogenic Agar Listeria Ottaviani Agosti agar (ALOA, 91% of isolates) and non-selective horse blood agar (HBA, 89%) for 24h. Chemometric statistical analysis of the MALDI-TOF MS data enabled source-tracking of L. monocytogenes isolates obtained from four different dairy sources. Strain-level discrimination was also observed to be influenced by culture conditions. In addition, t-test/analysis of variance (ANOVA) was used to identify potential biomarker peaks that differentiated the isolates according to their source of isolation. Source-tracking using MALDI-TOF MS was compared and correlated with the gold standard pulsed-field gel electrophoresis (PFGE) technique. The discriminatory index and the congruence between both techniques were compared using the Simpsons Diversity Index and adjusted Rand and Wallace coefficients. Overall, MALDI-TOF MS based source-tracking (using data obtained by culturing the isolates on HBA) and PFGE demonstrated good congruence with a Wallace coefficient of 0.71 and

  18. Identification of SCN1A and PCDH19 mutations in Chinese children with Dravet syndrome.

    Directory of Open Access Journals (Sweden)

    Anna Ka-Yee Kwong

    Full Text Available BACKGROUND: Dravet syndrome is a severe form of epilepsy. Majority of patients have a mutation in SCN1A gene, which encodes a voltage-gated sodium channel. A recent study has demonstrated that 16% of SCN1A-negative patients have a mutation in PCDH19, the gene encoding protocadherin-19. Mutations in other genes account for only a very small proportion of families. TSPYL4 is a novel candidate gene within the locus 6q16.3-q22.31 identified by linkage study. OBJECTIVE: The present study examined the mutations in epileptic Chinese children with emphasis on Dravet syndrome. METHODS: A hundred children with severe epilepsy were divided into Dravet syndrome and non-Dravet syndrome groups and screened for SCN1A mutations by direct sequencing. SCN1A-negative Dravet syndrome patients and patients with phenotypes resembling Dravet syndrome were checked for PCDH19 and TSPYL4 mutations. RESULTS: Eighteen patients (9 males, 9 females were diagnosed to have Dravet syndrome. Among them, 83% (15/18 had SCN1A mutations including truncating (7, splice site (2 and missense mutations (6. The truncating/splice site mutations were associated with moderate to severe degree of intellectual disability (p<0.05. During the progression of disease, 73% (11/15 had features fitting into the diagnostic criteria of autism spectrum disorder and 53% (8/15 had history of vaccination-induced seizures. A novel PCDH19 p.D377N mutation was identified in one SCN1A-negative female patient with Dravet syndrome and a known PCDH19 p.N340S mutation in a female non-Dravet syndrome patient. The former also inherited a TSPYL4 p.G60R variant. CONCLUSION: A high percentage of SCN1A mutations was identified in our Chinese cohort of Dravet syndrome patients but none in the rest of patients. We demonstrated that truncating/splice site mutations were linked to moderate to severe intellectual disability in these patients. A de novo PCDH19 missense mutation together with an inherited TSPYL4 missense

  19. Rapid and reliable detection and identification of GM events using multiplex PCR coupled with oligonucleotide microarray.

    Science.gov (United States)

    Xu, Xiaodan; Li, Yingcong; Zhao, Heng; Wen, Si-yuan; Wang, Sheng-qi; Huang, Jian; Huang, Kun-lun; Luo, Yun-bo

    2005-05-18

    To devise a rapid and reliable method for the detection and identification of genetically modified (GM) events, we developed a multiplex polymerase chain reaction (PCR) coupled with a DNA microarray system simultaneously aiming at many targets in a single reaction. The system included probes for screening gene, species reference gene, specific gene, construct-specific gene, event-specific gene, and internal and negative control genes. 18S rRNA was combined with species reference genes as internal controls to assess the efficiency of all reactions and to eliminate false negatives. Two sets of the multiplex PCR system were used to amplify four and five targets, respectively. Eight different structure genes could be detected and identified simultaneously for Roundup Ready soybean in a single microarray. The microarray specificity was validated by its ability to discriminate two GM maizes Bt176 and Bt11. The advantages of this method are its high specificity and greatly reduced false-positives and -negatives. The multiplex PCR coupled with microarray technology presented here is a rapid and reliable tool for the simultaneous detection of GM organism ingredients.

  20. Rapid detection and identification of four major Schistosoma species by high-resolution melt (HRM) analysis.

    Science.gov (United States)

    Li, Juan; Zhao, Guang-Hui; Lin, RuiQing; Blair, David; Sugiyama, Hiromu; Zhu, Xing-Quan

    2015-11-01

    Schistosomiasis, caused by blood flukes belonging to several species of the genus Schistosoma, is a serious and widespread parasitic disease. Accurate and rapid differentiation of these etiological agents of animal and human schistosomiasis to species level can be difficult. We report a real-time PCR assay coupled with a high-resolution melt (HRM) assay targeting a portion of the nuclear 18S rDNA to detect, identify, and distinguish between four major blood fluke species (Schistosoma japonicum, Schistosoma mansoni, Schistosoma haematobium, and Schistosoma mekongi). Using this system, the Schistosoma spp. was accurately identified and could also be distinguished from all other trematode species with which they were compared. As little as 10(-5) ng genomic DNA from a Schistosoma sp. could be detected. This process is inexpensive, easy, and can be completed within 3 h. Examination of 21 representative Schistosoma samples from 15 geographical localities in seven endemic countries validated the value of the HRM detection assay and proved its reliability. The melting curves were characterized by peaks of 83.65 °C for S. japonicum and S. mekongi, 85.65 °C for S. mansoni, and 85.85 °C for S. haematobium. The present study developed a real-time PCR coupled with HRM analysis assay for detection and differential identification of S. mansoni, S. haematobium, S. japonicum, and S. mekongi. This method is rapid, sensitive, and inexpensive. It has important implications for epidemiological studies of Schistosoma.

  1. [Evaluation of Prolex for the rapid identification of streptococci isolated in medical microbiology].

    Science.gov (United States)

    Loubinoux, J; Mihaila-Amrouche, L; Bouvet, A

    2004-10-01

    The need to rapidly identify streptococci responsible for acute infectious diseases has led to the development of agglutination techniques that are able to identify streptococcal group antigens (A, B, C, D, F, and G) directly from primoculture colonies on blood agar. The Prolex agglutination tests (Pro-Lab Diagnostics, Richmond Hill, Ontario, Canada), distributed in France by i2a, have been used for the determination of group antigens of 166 isolates of streptococci and enterococci previously identified in the National Reference Center for Streptococci. The results obtained with the Prolex reagents have permitted to correctly identify all pyogenic beta-hemolytic streptococci (23 Streptococcus pyogenes, 21 Streptococcus agalactiae, 33 Streptococcus dysgalactiae subsp. equisimilis including 6 group C and 27 group G, and 5 Streptococcus porcinus including 4 group B). Four differences between unexpected agglutinations (A or F) and species identifications have been obtained. These differences were observed for four non-hemolytic isolates of Streptococcus mutans, Streptococcus gordonii, Streptococcus infantarius, and Streptococcus suis. The anti-D reagent has been of value as a marker for isolates of enterococci. Thus, these results confirm the abilities of these agglutination tests for the grouping of beta-hemolytic streptococci. Moreover, the use of Prolex has the advantage to be rapid because of the non-enzymatic but chemical extraction of streptococcal antigens.

  2. Identification of novel mutations in Chinese Hans with autosomal dominant polycystic kidney disease

    Directory of Open Access Journals (Sweden)

    Yu Chaowen

    2011-12-01

    Full Text Available Abstract Background Autosomal dominant polycystic kidney disease (ADPKD is the most common inherited renal disease with an incidence of 1 in 400 to 1000. The disease is genetically heterogeneous, with two genes identified: PKD1 (16p13.3 and PKD2 (4q21. Molecular diagnosis of the disease in at-risk individuals is complicated due to the structural complexity of PKD1 gene and the high diversity of the mutations. This study is the first systematic ADPKD mutation analysis of both PKD1 and PKD2 genes in Chinese patients using denaturing high-performance liquid chromatography (DHPLC. Methods Both PKD1 and PKD2 genes were mutation screened in each proband from 65 families using DHPLC followed by DNA sequencing. Novel variations found in the probands were checked in their family members available and 100 unrelated normal controls. Then the pathogenic potential of the variations of unknown significance was examined by evolutionary comparison, effects of amino acid substitutions on protein structure, and effects of splice site alterations using online mutation prediction resources. Results A total of 92 variations were identified, including 27 reported previously. Definitely pathogenic mutations (ten frameshift, ten nonsense, two splicing defects and one duplication were identified in 28 families, and probably pathogenic mutations were found in an additional six families, giving a total detection level of 52.3% (34/65. About 69% (20/29 of the mutations are first reported with a recurrent mutation rate of 31%. Conclusions Mutation study of PKD1 and PKD2 genes in Chinese Hans with ADPKD may contribute to a better understanding of the genetic diversity between different ethnic groups and enrich the mutation database. Besides, evaluating the pathogenic potential of novel variations should also facilitate the clinical diagnosis and genetic counseling of the disease.

  3. Identification of a Non-Gatekeeper Hot Spot for Drug-Resistant Mutations in mTOR Kinase.

    Science.gov (United States)

    Wu, Tzung-Ju; Wang, Xiaowen; Zhang, Yanjie; Meng, Linghua; Kerrigan, John E; Burley, Stephen K; Zheng, X F Steven

    2015-04-21

    Protein kinases are therapeutic targets for human cancer. However, "gatekeeper" mutations in tyrosine kinases cause acquired clinical resistance, limiting long-term treatment benefits. mTOR is a key cancer driver and drug target. Numerous small-molecule mTOR kinase inhibitors have been developed, with some already in human clinical trials. Given our clinical experience with targeted therapeutics, acquired drug resistance in mTOR is thought likely, but not yet documented. Herein, we describe identification of a hot spot (L2185) for drug-resistant mutations, which is distinct from the gatekeeper site, and a chemical scaffold refractory to drug-resistant mutations. We also provide new insights into mTOR kinase structure and function. The hot spot mutations are potentially useful as surrogate biomarkers for acquired drug resistance in ongoing clinical trials and future treatments and for the design of the next generation of mTOR-targeted drugs. Our study provides a foundation for further research into mTOR kinase function and targeting. Copyright © 2015 The Authors. Published by Elsevier Inc. All rights reserved.

  4. [Automated RNA amplification for the rapid identification of Mycobacterium tuberculosis complex in respiratory specimens].

    Science.gov (United States)

    Drouillon, V; Houriez, F; Buze, M; Lagrange, P; Herrmann, J-L

    2006-01-01

    Rapid and sensitive detection of Mycobacterium tuberculosis complex (MTB) directly on clinical respiratory specimens is essential for a correct management of patients suspected of tuberculosis. For this purpose PCR-based kits are available to detect MTB in respiratory specimen but most of them need at least 4 hours to be completed. New methods, based on TRC method (TRC: Transcription Reverse transcription Concerted--TRCRapid M. Tuberculosis--Tosoh Bioscience, Tokyo, Japon) and dedicated monitor have been developed. A new kit (TRC Rapid M. tuberculosis and Real-time monitor TRCRapid-160, Tosoh Corporation, Japan) enabling one step amplification and real-time detection of MTB 16S rRNA by a combination of intercalative dye oxazole yellow-linked DNA probe and isothermal RNA amplification directly on respiratory specimens has been tested in our laboratory. 319 respiratory specimens were tested in this preliminary study and results were compared to smear and culture. Fourteen had a positive culture for MTB. Among theses samples, smear was positive in 11 cases (78.6%) and TRC process was positive in 8 cases (57.1%). Overall sensitivity of TRC compared to smear positive samples is 73%. Theses first results demonstrated that a rapid identification of MTB was possible (less than 2 processing hours for 14 specimens and about 1 hour for 1 specimen) in most cases of smear positive samples using ready to use reagents for real time detection of MTB rRNA in clinical samples. New pretreatment and extraction reagents kits to increase the stability of the sputum RNA and the extraction efficiency are now tested in our laboratory.

  5. Identification of novel mutations in HEXA gene in children affected with Tay Sachs disease from India.

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    Mehul Mistri

    Full Text Available Tay Sachs disease (TSD is a neurodegenerative disorder due to β-hexosaminidase A deficiency caused by mutations in the HEXA gene. The mutations leading to Tay Sachs disease in India are yet unknown. We aimed to determine mutations leading to TSD in India by complete sequencing of the HEXA gene. The clinical inclusion criteria included neuroregression, seizures, exaggerated startle reflex, macrocephaly, cherry red spot on fundus examination and spasticity. Neuroimaging criteria included thalamic hyperdensities on CT scan/T1W images of MRI of the brain. Biochemical criteria included deficiency of hexosaminidase A (less than 2% of total hexosaminidase activity for infantile patients. Total leukocyte hexosaminidase activity was assayed by 4-methylumbelliferyl-N-acetyl-β-D-glucosamine lysis and hexosaminidase A activity was assayed by heat inactivation method and 4-methylumbelliferyl-N-acetyl-β-D-glucosamine-6-sulphate lysis method. The exons and exon-intron boundaries of the HEXA gene were bidirectionally sequenced using an automated sequencer. Mutations were confirmed in parents and looked up in public databases. In silico analysis for mutations was carried out using SIFT, Polyphen2, MutationT@ster and Accelrys Discovery Studio softwares. Fifteen families were included in the study. We identified six novel missense mutations, c.340 G>A (p.E114K, c.964 G>A (p.D322N, c.964 G>T (p.D322Y, c.1178C>G (p.R393P and c.1385A>T (p.E462V, c.1432 G>A (p.G478R and two previously reported mutations. c.1277_1278insTATC and c.508C>T (p.R170W. The mutation p.E462V was found in six unrelated families from Gujarat indicating a founder effect. A previously known splice site mutation c.805+1 G>C and another intronic mutation c.672+30 T>G of unknown significance were also identified. Mutations could not be identified in one family. We conclude that TSD patients from Gujarat should be screened for the common mutation p.E462V.

  6. Identification of novel mutations in HEXA gene in children affected with Tay Sachs disease from India.

    Science.gov (United States)

    Mistri, Mehul; Tamhankar, Parag M; Sheth, Frenny; Sanghavi, Daksha; Kondurkar, Pratima; Patil, Swapnil; Idicula-Thomas, Susan; Gupta, Sarita; Sheth, Jayesh

    2012-01-01

    Tay Sachs disease (TSD) is a neurodegenerative disorder due to β-hexosaminidase A deficiency caused by mutations in the HEXA gene. The mutations leading to Tay Sachs disease in India are yet unknown. We aimed to determine mutations leading to TSD in India by complete sequencing of the HEXA gene. The clinical inclusion criteria included neuroregression, seizures, exaggerated startle reflex, macrocephaly, cherry red spot on fundus examination and spasticity. Neuroimaging criteria included thalamic hyperdensities on CT scan/T1W images of MRI of the brain. Biochemical criteria included deficiency of hexosaminidase A (less than 2% of total hexosaminidase activity for infantile patients). Total leukocyte hexosaminidase activity was assayed by 4-methylumbelliferyl-N-acetyl-β-D-glucosamine lysis and hexosaminidase A activity was assayed by heat inactivation method and 4-methylumbelliferyl-N-acetyl-β-D-glucosamine-6-sulphate lysis method. The exons and exon-intron boundaries of the HEXA gene were bidirectionally sequenced using an automated sequencer. Mutations were confirmed in parents and looked up in public databases. In silico analysis for mutations was carried out using SIFT, Polyphen2, MutationT@ster and Accelrys Discovery Studio softwares. Fifteen families were included in the study. We identified six novel missense mutations, c.340 G>A (p.E114K), c.964 G>A (p.D322N), c.964 G>T (p.D322Y), c.1178C>G (p.R393P) and c.1385A>T (p.E462V), c.1432 G>A (p.G478R) and two previously reported mutations. c.1277_1278insTATC and c.508C>T (p.R170W). The mutation p.E462V was found in six unrelated families from Gujarat indicating a founder effect. A previously known splice site mutation c.805+1 G>C and another intronic mutation c.672+30 T>G of unknown significance were also identified. Mutations could not be identified in one family. We conclude that TSD patients from Gujarat should be screened for the common mutation p.E462V.

  7. Identification of a nuclear localization signal in the retinitis pigmentosa-mutated RP26 protein, ceramide kinase-like protein

    International Nuclear Information System (INIS)

    Inagaki, Yuichi; Mitsutake, Susumu; Igarashi, Yasuyuki

    2006-01-01

    Retinitis pigmentosa (RP) is a genetically heterogeneous disease characterized by degeneration of the retina. A mutation in a new ceramide kinase (CERK) homologous gene, named CERK-like protein (CERKL), was found to cause autosomal recessive retinitis pigmentosa (RP26). Here, we show a point mutation of one of two putative nuclear localization signal (NLS) sequences inhibited the nuclear localization of the protein. Furthermore, the tetra-GFP-tagged NLS, which cannot passively enter the nucleus, was observed not only in the nucleus but also in the nucleolus. Our results provide First evidence of the active nuclear import of CERKL and suggest that the identified NLS might be responsible for nucleolar retention of the protein. As recent studies have shown other RP-related proteins are localized in the nucleus or the nucleolus, our identification of NLS in CERKL suggests that CERKL likely plays important roles for retinal functions in the nucleus and the nucleolus

  8. Identification of two novel pathogenic compound heterozygous MYO7A mutations in Usher syndrome by whole exome sequencing.

    Science.gov (United States)

    Jia, Ying; Li, Xiaoge; Yang, Dong; Xu, Yi; Guo, Ying; Li, Xin

    2018-01-01

    The current study aims to identify the pathogenic sites in a core pedigree of Usher syndrome (USH). A core pedigree of USH was analyzed by whole exome sequencing (WES). Mutations were verified by polymerase chain reaction (PCR) amplification and Sanger sequencing. Two pathogenic variations (c.849+2T>C and c.5994G>A) in MYO7A were successfully identified and individually separated from parents. One variant (c.849+2T>C) was nonsense mutation, causing the protein terminated in advance, and the other one (c.5994G>A) located near the boundary of exon could cause aberrant splicing. This study provides a meaningful exploration for identification of clinical core genetic pedigrees. Copyright © 2017 Elsevier B.V. All rights reserved.

  9. Identification and functional analysis of SOX10 missense mutations in different subtypes of Waardenburg syndrome.

    Science.gov (United States)

    Chaoui, Asma; Watanabe, Yuli; Touraine, Renaud; Baral, Viviane; Goossens, Michel; Pingault, Veronique; Bondurand, Nadege

    2011-12-01

    Waardenburg syndrome (WS) is a rare disorder characterized by pigmentation defects and sensorineural deafness, classified into four clinical subtypes, WS1-S4. Whereas the absence of additional features characterizes WS2, association with Hirschsprung disease defines WS4. WS is genetically heterogeneous, with six genes already identified, including SOX10. About 50 heterozygous SOX10 mutations have been described in patients presenting with WS2 or WS4, with or without myelination defects of the peripheral and central nervous system (PCWH, Peripheral demyelinating neuropathy-Central dysmyelinating leukodystrophy-Waardenburg syndrome-Hirschsprung disease, or PCW, PCWH without HD). The majority are truncating mutations that most often remove the main functional domains of the protein. Only three missense mutations have been thus far reported. In the present study, novel SOX10 missense mutations were found in 11 patients and were examined for effects on SOX10 characteristics and functions. The mutations were associated with various phenotypes, ranging from WS2 to PCWH. All tested mutations were found to be deleterious. Some mutants presented with partial cytoplasmic redistribution, some lost their DNA-binding and/or transactivation capabilities on various tissue-specific target genes. Intriguingly, several mutants were redistributed in nuclear foci. Whether this phenomenon is a cause or a consequence of mutation-associated pathogenicity remains to be determined, but this observation could help to identify new SOX10 modes of action. © 2011 Wiley-Liss, Inc.

  10. Identification of five novel FBN1 mutations by non-radioactive single-strand conformation analysis

    Energy Technology Data Exchange (ETDEWEB)

    Liu, W.; Qian, C.; Comeau, K.; Francke, U. [Stanford Univ. Medical Center, Stanford, CA (United States)

    1994-09-01

    Marfan syndrome (MFS), one of the most common genetic disorders of connective tissue, is characterized by variable manifestations in skeletal, cardiovascular and ocular systems. Mutations in the fibrillin gene on chromosome 15 (FBN1) have been shown to cause MFS. To examine the relationship between FBN1 gene mutations, fibrillin protein function and MFS phenotypes, we screened for alternations in the fibrillin coding sequence in fibroblast derived cDNA from MFS patients. To date, abnormally migrating bands in more than 20 unrelated MFS patients have been identified by using non-radioactive single-strand conformation analysis and silver staining. Five altered bands have been directly sequenced. Two missense mutations and three splice site mutations have been identified. Both missense mutations substitute another amino acid for a cysteine residue (C1402W and C1672R) in EGF-like motifs of the fibrillin polypeptide chain. The two splice site mutations are at nucleotide positions 6994+1 (G{yields}A), and 7205-2 (A{yields}G) and result in in-frame skipping of exon 56 and 58, respectively. Skipping of exon 56 occurs in 50% of mutant transcripts. Use of a cryptic splice site 51 bp upstream of the normal donor site results in half of the mutant transcripts containing part of exon 56. Both products contain in-frame deletions. Another splice site mutation, identified by exon screening from patient genomic DNA using intron primers, is at nucleotide position 2293+2 (T{yields}A), but the predicted exon skipping has not been detected at the RT-PCR level. This may be due to instability of the mutant transcript. Including the mutations reported here, a total of 8 out of 36 published FBN1 gene mutations involve exon skipping. It may be inferred that FBN1 exon skipping plays an important pathogenic role in MFS.

  11. Rapidly progressive renal disease as part of Wolfram syndrome in a large inbred Turkish family due to a novel WFS1 mutation (p.Leu511Pro)

    DEFF Research Database (Denmark)

    Yuca, Sevil Ari; Rendtorff, Nanna Dahl; Boulahbel, Houda

    2012-01-01

    Wolfram syndrome, also named "DIDMOAD" (diabetes insipidus, diabetes mellitus, optic atrophy, and deafness), is an inherited association of juvenile-onset diabetes mellitus and optic atrophy as key diagnostic criteria. Renal tract abnormalities and neurodegenerative disorder may occur in the third...... and fourth decade. The wolframin gene, WFS1, associated with this syndrome, is located on chromosome 4p16.1. Many mutations have been described since the identification of WFS1 as the cause of Wolfram syndrome. We identified a new homozygous WFS1 mutation (c.1532T>C; p.Leu511Pro) causing Wolfram syndrome...

  12. Molecular genetic analysis of the F11 gene in 14 Turkish patients with factor XI deficiency: identification of novel and recurrent mutations and their inheritance within families.

    Science.gov (United States)

    Colakoglu, Seyma; Bayhan, Turan; Tavil, Betül; Keskin, Ebru Yılmaz; Cakir, Volkan; Gümrük, Fatma; Çetin, Mualla; Aytaç, Selin; Berber, Ergul

    2018-01-01

    Factor XI (FXI) deficiency is an autosomal bleeding disease associated with genetic defects in the F11 gene which cause decreased FXI levels or impaired FXI function. An increasing number of mutations has been reported in the FXI mutation database, most of which affect the serine protease domain of the protein. FXI is a heterogeneous disorder associated with a variable bleeding tendency and a variety of causative F11 gene mutations. The molecular basis of FXI deficiency in 14 patients from ten unrelated families in Turkey was analysed to establish genotype-phenotype correlations and inheritance of the mutations in the patients' families. Fourteen index cases with a diagnosis of FXI deficiency and family members of these patients were enrolled into the study. The patients' F11 genes were amplified by polymerase chain reaction and subjected to direct DNA sequencing analysis. The findings were analysed statistically using bivariate correlations, Pearson's correlation coefficient and the nonparametric Mann-Whitney test. Direct DNA sequencing analysis of the F11 genes revealed that all of the 14 patients had a F11 gene mutation. Eight different mutations were identified in the apple 1, apple 2 or serine protease domains, except one which was a splice site mutation. Six of the mutations were recurrent. Two of the mutations were novel missense mutations, p.Val522Gly and p.Cys581Arg, within the catalytic domain. The p.Trp519Stop mutation was observed in two families whereas all the other mutations were specific to a single family. Identification of mutations confirmed the genetic heterogeneity of FXI deficiency. Most of the patients with mutations did not have any bleeding complications, whereas some had severe bleeding symptoms. Genetic screening for F11 gene mutations is important to decrease the mortality and morbidity rate associated with FXI deficiency, which can be life-threatening if bleeding occurs in tissues with high fibrinolytic activity.

  13. Identification of three novel NHS mutations in families with Nance-Horan syndrome.

    Science.gov (United States)

    Huang, Kristen M; Wu, Junhua; Brooks, Simon P; Hardcastle, Alison J; Lewis, Richard Alan; Stambolian, Dwight

    2007-03-27

    Nance-Horan Syndrome (NHS) is an infrequent and often overlooked X-linked disorder characterized by dense congenital cataracts, microphthalmia, and dental abnormalities. The syndrome is caused by mutations in the NHS gene, whose function is not known. The purpose of this study was to identify the frequency and distribution of NHS gene mutations and compare genotype with Nance-Horan phenotype in five North American NHS families. Genomic DNA was isolated from white blood cells from NHS patients and family members. The NHS gene coding region and its splice site donor and acceptor regions were amplified from genomic DNA by PCR, and the amplicons were sequenced directly. We identified three unique NHS coding region mutations in these NHS families. This report extends the number of unique identified NHS mutations to 14.

  14. Genotype-Phenotype Correlations Emerging from the Identification of Missense Mutations in MBTPS2

    NARCIS (Netherlands)

    Bornholdt, D.; Atkinson, T.P.; Bouadjar, B.; Catteau, B.; Cox, H.; Silva, D. De; Fischer, J.; Gunasekera, C.N.; Hadj-Rabia, S.; Happle, R.; Holder-Espinasse, M.; Kaminski, E.; Konig, A.; Megarbane, A.; Megarbane, H.; Neidel, U.; Oeffner, F.; Oji, V.; Theos, A.; Traupe, H.; Vahlquist, A.; Bon, B.W. van; Virtanen, M.; Grzeschik, K.H.

    2013-01-01

    Missense mutations affecting membrane-bound transcription factor protease site 2 (MBTPS2) have been associated with Ichthyosis Follicularis with Atrichia and Photophobia (IFAP) syndrome with or without BRESHECK syndrome, with keratosis follicularis spinulosa decalvans, and Olmsted syndrome. This

  15. A second mutation associated with apparent [beta]-hexosaminidase A pseudodeficiency: Identification and frequency estimation

    Energy Technology Data Exchange (ETDEWEB)

    Cao, Z.; Chabot, T.; Triggs-Raine, B.L. (Univ. of Manitoba, Winnepeg (Canada)); Natowicz, M.R.; Prence, E.M. (Shriver Center for Mental Retardation, Waltham, MA (United States) Harvard Medical School, Boston, MA (United States)); Kaback, M.M.; Lim-Steele, S.T.; Brown, D. (Children' s Hospital, San Diego, CA (United States) Univ. of California, San Diego, CA (United States))

    1993-12-01

    Deficient activity of [beta]-hexosaminidase A (Hex A), resulting from mutations in the HEXA gene, typically causes Tay-Sachs disease. However, healthy individuals lacking Hex A activity against synthetic substrates (i.e., individuals who are pseudodeficient) have been described. Recently, an apparently benign C[sub 739]-to-T (Arg247Trp) mutation was found among individuals with Hex A levels indistinguishable from those of carriers of Tay-Sachs disease. This allele, when in compound heterozygosity with a second [open quotes]disease-causing[close quotes] allele, results in Hex A pseudodeficiency. The authors examined the HEXA gene of a healthy 42-year-old who was Hex A deficient but did not have the C[sub 739]-to-T mutation. The HEXA exons were PCR amplified, and the products were analyzed for mutations by using restriction-enzyme digestion or single-strand gel electrophoresis. A G[sub 805]-to-A (Gly269Ser) mutation associated with adult-onset G[sub m2] gangliosidosis was found on one chromosome. A new mutation, C[sub 745]-to-T (Arg 249Trp), was identified on the second chromosome. This mutation was detected in an additional 4/63 (6%) non-Jewish and 0/218 Ashkenazi Jewish enzyme-defined carriers. Although the Arg249Trp change may result in a late-onset form of G[sub M2] gangliosidosis, any phenotype must be very mild. This new mutation and the benign C[sub 739]-to-T mutation together account for [approximately]38% of non-Jewish enzyme-defined carriers. Because carriers of the C[sub 739]-to-T and C[sub 745]-to-T mutations cannot be differentiated from carriers of disease-causing alleles by using the classical biochemical screening approaches, DNA-based analyses for these mutations should be offered for non-Jewish enzyme-defined heterozygotes, before definitive counseling is provided. 46 refs., 5 figs., 2 tabs.

  16. Identification of three novel NHS mutations in families with Nance-Horan syndrome

    OpenAIRE

    Huang, Kristen M.; Wu, Junhua; Brooks, Simon P.; Hardcastle, Alison J.; Lewis, Richard Alan; Stambolian, Dwight

    2007-01-01

    Purpose Nance-Horan Syndrome (NHS) is an infrequent and often overlooked X-linked disorder characterized by dense congenital cataracts, microphthalmia, and dental abnormalities. The syndrome is caused by mutations in the NHS gene, whose function is not known. The purpose of this study was to identify the frequency and distribution of NHS gene mutations and compare genotype with Nance-Horan phenotype in five North American NHS families. Methods Genomic DNA was isolated from white blood cells f...

  17. Identification of 3 novel VHL germ-line mutations in Danish VHL patients

    DEFF Research Database (Denmark)

    Dandanell, Mette; Friis-Hansen, Lennart Jan; Sunde, Lone

    2012-01-01

    von Hippel-Lindau (VHL) disease is a hereditary cancer syndrome in which the patients develop retinal and central nervous system hemangioblastomas, pheochromocytomas and clear-cell renal tumors. The autosomal dominant disease is caused by mutations in the VHL gene.......von Hippel-Lindau (VHL) disease is a hereditary cancer syndrome in which the patients develop retinal and central nervous system hemangioblastomas, pheochromocytomas and clear-cell renal tumors. The autosomal dominant disease is caused by mutations in the VHL gene....

  18. Identification of functional mutations in GATA4 in patients with congenital heart disease.

    Directory of Open Access Journals (Sweden)

    Erli Wang

    Full Text Available Congenital heart disease (CHD is one of the most prevalent developmental anomalies and the leading cause of noninfectious morbidity and mortality in newborns. Despite its prevalence and clinical significance, the etiology of CHD remains largely unknown. GATA4 is a highly conserved transcription factor that regulates a variety of physiological processes and has been extensively studied, particularly on its role in heart development. With the combination of TBX5 and MEF2C, GATA4 can reprogram postnatal fibroblasts into functional cardiomyocytes directly. In the past decade, a variety of GATA4 mutations were identified and these findings originally came from familial CHD pedigree studies. Given that familial and sporadic CHD cases allegedly share a basic genetic basis, we explore the GATA4 mutations in different types of CHD. In this study, via direct sequencing of the GATA4 coding region and exon-intron boundaries in 384 sporadic Chinese CHD patients, we identified 12 heterozygous non-synonymous mutations, among which 8 mutations were only found in CHD patients when compared with 957 controls. Six of these non-synonymous mutations have not been previously reported. Subsequent functional analyses revealed that the transcriptional activity, subcellular localization and DNA binding affinity of some mutant GATA4 proteins were significantly altered. Our results expand the spectrum of GATA4 mutations linked to cardiac defects. Together with the newly reported mutations, approximately 110 non-synonymous mutations have currently been identified in GATA4. Our future analysis will explore why the evolutionarily conserved GATA4 appears to be hypermutable.

  19. Rapid identification of fluorochrome modification sites in proteins by LC ESI-Q-TOF mass spectrometry.

    Science.gov (United States)

    Manikwar, Prakash; Zimmerman, Tahl; Blanco, Francisco J; Williams, Todd D; Siahaan, Teruna J

    2011-07-20

    Conjugation of either a fluorescent dye or a drug molecule to the ε-amino groups of lysine residues of proteins has many applications in biology and medicine. However, this type of conjugation produces a heterogeneous population of protein conjugates. Because conjugation of fluorochrome or drug molecule to a protein may have deleterious effects on protein function, the identification of conjugation sites is necessary. Unfortunately, the identification process can be time-consuming and laborious; therefore, there is a need to develop a rapid and reliable way to determine the conjugation sites of the fluorescent label or drug molecule. In this study, the sites of conjugation of fluorescein-5'-isothiocyanate and rhodamine-B-isothiocyanate to free amino groups on the insert-domain (I-domain) protein derived from the α-subunit of lymphocyte function-associated antigen-1 (LFA-1) were determined by electrospray ionization quadrupole time-of-flight mass spectrometry (ESI-Q-TOF MS) along with peptide mapping using trypsin digestion. A reporter fragment of the fluorochrome moiety that is generated in the collision cell of the Q-TOF without explicit MS/MS precursor selection was used to identify the conjugation site. Selected ion plots of the reporter ion readily mark modified peptides in chromatograms of the complex digest. Interrogation of theses spectra reveals a neutral loss/precursor pair that identifies the modified peptide. The results show that one to seven fluorescein molecules or one to four rhodamine molecules were attached to the lysine residue(s) of the I-domain protein. No modifications were found in the metal ion-dependent adhesion site (MIDAS), which is an important binding region of the I-domain.

  20. Rapid identification of bacteria and candida using pna-fish from blood and peritoneal fluid cultures: a retrospective clinical study

    Directory of Open Access Journals (Sweden)

    Harris Dana M

    2013-01-01

    Full Text Available Abstract Background Peptide nucleic acid fluorescent in situ hybridization (PNA-FISH is a rapid and established method for identification of Candida sp., Gram positive, and Gram negative bacteria from positive blood cultures. This study reports clinical experience in the evaluation of 103 positive blood cultures and 17 positive peritoneal fluid cultures from 120 patients using PNA-FISH. Our study provides evidence as to potential pharmaceutical cost savings based on rapid pathogen identification, in addition to the novel application of PNA-FISH to peritoneal fluid specimens. Methods Identification accuracy and elapsed time to identification of Gram positives, Gram negatives, and Candida sp., isolated from blood and peritoneal fluid cultures were assessed using PNA-FISH (AdvanDx, as compared to standard culture methods. Patient charts were reviewed to extrapolate potential pharmaceutical cost savings due to adjustment of antimicrobial or antifungal therapy, based on identification by PNA-FISH. Results In blood cultures, time to identification by standard culture methods for bacteria and Candida sp., averaged 83.6 hours (95% CI 56.7 to 110.5. Identification by PNA-FISH averaged 11.2 hours (95% CI 4.8 to 17.6. Overall PNA-FISH identification accuracy was 98.8% (83/84, 95% CI 93.5% to 99.9% as compared to culture. In peritoneal fluid, identification of bacteria by culture averaged 87.4 hours (95% CI −92.4 to 267.1. Identification by PNA-FISH averaged 16.4 hours (95% CI −57.3 to 90.0. Overall PNA-FISH identification accuracy was 100% (13/13, 95% CI 75.3% to 100%. For Candida sp., pharmaceutical cost savings based on PNA-FISH identification could be $377.74/day. For coagulase-negative staphylococcus (CoNS, discontinuation of vancomycin could result in savings of $20.00/day. Conclusions In this retrospective study, excellent accuracy of PNA-FISH in blood and peritoneal fluids with reduced time to identification was observed, as compared to

  1. Identification of Two Novel HOXB13 Germline Mutations in Portuguese Prostate Cancer Patients

    Science.gov (United States)

    Maia, Sofia; Cardoso, Marta; Pinto, Pedro; Pinheiro, Manuela; Santos, Catarina; Peixoto, Ana; Bento, Maria José; Oliveira, Jorge; Henrique, Rui; Jerónimo, Carmen; Teixeira, Manuel R.

    2015-01-01

    The HOXB13 germline variant G84E (rs138213197) was recently described in men of European descent, with the highest prevalence in Northern Europe. The G84E mutation has not been found in patients of African or Asian ancestry, which may carry other HOXB13 variants, indicating allelic heterogeneity depending on the population. In order to gain insight into the full scope of coding HOXB13 mutations in Portuguese prostate cancer patients, we decided to sequence the entire coding region of the HOXB13 gene in 462 early-onset or familial/hereditary cases. Additionally, we searched for somatic HOXB13 mutations in 178 prostate carcinomas to evaluate their prevalence in prostate carcinogenesis. Three different patients were found to carry in their germline DNA two novel missense variants, which were not identified in 132 control subjects. Both variants are predicted to be deleterious by different in silico tools. No somatic mutations were found. These findings further support the hypothesis that different rare HOXB13 mutations may be found in different ethnic groups. Detection of mutations predisposing to prostate cancer may require re-sequencing rather than genotyping, as appropriate to the population under investigation. PMID:26176944

  2. Identification of a novel WFS1 homozygous nonsense mutation in Jordanian children with Wolfram syndrome.

    Science.gov (United States)

    Bodoor, Khaldon; Batiha, Osama; Abu-Awad, Ayman; Al-Sarihin, Khaldon; Ziad, Haya; Jarun, Yousef; Abu-Sheikha, Aya; Abu Jalboush, Sara; Alibrahim, Khoulod S

    2016-09-01

    Wolfram syndrome (WS) is a rare autosomal recessive neurodegenerative disorder characterized by the presentation of early onset type I diabetes mellitus and optic atrophy with later onset diabetes insipidus and deafness. WFS1 gene was identified on chromosome 4p16.1 as the gene responsible for WS disease given that most of the WS patients were found to carry mutations in this gene. This study was carried out to investigate the molecular spectrum of WFS1 gene in Jordanian families. Molecular and clinical characterization was performed on five WS patients from two unrelated Jordanian families. Our data indicated that WS patients of the first family harbored two deletion mutations (V415del and F247fs) located in exon 8 and exon 7 respectively, with a compound heterozygous pattern of inheritance; while in the second family, we identified a novel nonsense mutation (W185X) located in exon 5 in the N-terminal cytoplasmic domain with a homozygous pattern of inheritance. This mutation can be considered as loss of function mutation since the resulting truncated protein lost both the transmembrane domain and the C-terminal domain. Additionally, the W185X mutation lies within the CaM binding domain in wolframin protein which is thought to have a role in the regulation of wolframin function in response to calcium levels.

  3. Improved method for rapid and accurate isolation and identification of Streptococcus mutans and Streptococcus sobrinus from human plaque samples.

    Science.gov (United States)

    Villhauer, Alissa L; Lynch, David J; Drake, David R

    2017-08-01

    Mutans streptococci (MS), specifically Streptococcus mutans (SM) and Streptococcus sobrinus (SS), are bacterial species frequently targeted for investigation due to their role in the etiology of dental caries. Differentiation of S. mutans and S. sobrinus is an essential part of exploring the role of these organisms in disease progression and the impact of the presence of either/both on a subject's caries experience. Of vital importance to the study of these organisms is an identification protocol that allows us to distinguish between the two species in an easy, accurate, and timely manner. While conducting a 5-year birth cohort study in a Northern Plains American Indian tribe, the need for a more rapid procedure for isolating and identifying high volumes of MS was recognized. We report here on the development of an accurate and rapid method for MS identification. Accuracy, ease of use, and material and time requirements for morphological differentiation on selective agar, biochemical tests, and various combinations of PCR primers were compared. The final protocol included preliminary identification based on colony morphology followed by PCR confirmation of species identification using primers targeting regions of the glucosyltransferase (gtf) genes of SM and SS. This method of isolation and identification was found to be highly accurate, more rapid than the previous methodology used, and easily learned. It resulted in more efficient use of both time and material resources. Copyright © 2017 Elsevier B.V. All rights reserved.

  4. Rapid detection and identification of Bacillus anthracis in food using pyrosequencing technology.

    Science.gov (United States)

    Amoako, Kingsley K; Janzen, Timothy W; Shields, Michael J; Hahn, Kristen R; Thomas, Matthew C; Goji, Noriko

    2013-08-01

    The development of advanced methodologies for the detection of Bacillus anthracis has been evolving rapidly since the release of the anthrax spores in the mail in 2001. Recent advances in detection and identification techniques could prove to be an essential component in the defense against biological attacks. Sequence based such as pyrosequencing, which has the capability to determine short DNA stretches in real-time using biotinylated PCR amplicons, has potential biodefense applications. Using markers from the virulence plasmids (pXO1 and pXO2) and chromosomal regions, we have demonstrated the power of this technology in the rapid, specific and sensitive detection of B. anthracis spores in food matrices including milk, juice, bottled water, and processed meat. The combined use of immunomagnetic separation and pyrosequencing showed positive detection when liquid foods (bottled water, milk, juice), and processed meat were experimentally inoculated with 6CFU/mL and 6CFU/g, respectively, without an enrichment step. Pyrosequencing is completed in about 60min (following PCR amplification) and yields accurate and reliable results with an added layer of confidence. The entire assay (from sample preparation to sequencing information) can be completed in about 7.5h. A typical run on food samples yielded 67-80bp reads with 94-100% identity to the expected sequence. This sequence based approach is a novel application for the detection of anthrax spores in food with potential application in foodborne bioterrorism response and biodefense involving the use of anthrax spores. Crown Copyright © 2013. Published by Elsevier B.V. All rights reserved.

  5. Identification of a Variety of Mutations in Cancer Predisposition Genes in Patients With Suspected Lynch Syndrome.

    Science.gov (United States)

    Yurgelun, Matthew B; Allen, Brian; Kaldate, Rajesh R; Bowles, Karla R; Judkins, Thaddeus; Kaushik, Praveen; Roa, Benjamin B; Wenstrup, Richard J; Hartman, Anne-Renee; Syngal, Sapna

    2015-09-01

    Multigene panels are commercially available tools for hereditary cancer risk assessment that allow for next-generation sequencing of numerous genes in parallel. However, it is not clear if these panels offer advantages over traditional genetic testing. We investigated the number of cancer predisposition gene mutations identified by parallel sequencing in individuals with suspected Lynch syndrome. We performed germline analysis with a 25-gene, next-generation sequencing panel using DNA from 1260 individuals who underwent clinical genetic testing for Lynch syndrome from 2012 through 2013. All patients had a history of Lynch syndrome-associated cancer and/or polyps. We classified all identified germline alterations for pathogenicity and calculated the frequencies of pathogenic mutations and variants of uncertain clinical significance (VUS). We also analyzed data on patients' personal and family history of cancer, including fulfillment of clinical guidelines for genetic testing. Of the 1260 patients, 1112 met National Comprehensive Cancer Network (NCCN) criteria for Lynch syndrome testing (88%; 95% confidence interval [CI], 86%-90%). Multigene panel testing identified 114 probands with Lynch syndrome mutations (9.0%; 95% CI, 7.6%-10.8%) and 71 with mutations in other cancer predisposition genes (5.6%; 95% CI, 4.4%-7.1%). Fifteen individuals had mutations in BRCA1 or BRCA2; 93% of these met the NCCN criteria for Lynch syndrome testing and 33% met NCCN criteria for BRCA1 and BRCA2 analysis (P = .0017). An additional 9 individuals carried mutations in other genes linked to high lifetime risks of cancer (5 had mutations in APC, 3 had bi-allelic mutations in MUTYH, and 1 had a mutation in STK11); all of these patients met NCCN criteria for Lynch syndrome testing. A total of 479 individuals had 1 or more VUS (38%; 95% CI, 35%-41%). In individuals with suspected Lynch syndrome, multigene panel testing identified high-penetrance mutations in cancer predisposition genes, many

  6. The evidence of the rugoscopy effectiveness as a human identification method in patients submitted to rapid palatal expansion.

    Science.gov (United States)

    Barbieri, Ana A; Scoralick, Raquel A; Naressi, Suely C M; Moraes, Mari E L; Daruge, Eduardo; Daruge, Eduardo

    2013-01-01

    The objective of this study was to demonstrate the effectiveness of rugoscopy as a human identification method, even when the patient is submitted to rapid palatal expansion, which in theory would introduce doubt. With this intent, the Rugoscopic Identity was obtained for each subject using the classification formula proposed by Santos based on the intra-oral casts made before and after treatment from patients who were subjected to palatal expansion. The casts were labeled with the patients' initials and randomly arranged for studying. The palatine rugae kept the same patterns in every case studied. The technical error of the intra-evaluator measurement provided a confidence interval of 95%, making rugoscopy a reliable identification method for patients who were submitted to rapid palatal expansion, because even in the presence of intra-oral changes owing to the use of palatal expanders, the palatine rugae retained the biological and technical requirements for the human identification process. © 2012 American Academy of Forensic Sciences.

  7. Rapid detection of ERG11 gene mutations in clinical Candida albicans isolates with reduced susceptibility to fluconazole by rolling circle amplification and DNA sequencing

    Directory of Open Access Journals (Sweden)

    Ellis David

    2009-08-01

    Full Text Available Abstract Background Amino acid substitutions in the target enzyme Erg11p of azole antifungals contribute to clinically-relevant azole resistance in Candida albicans. A simple molecular method for rapid detection of ERG11 gene mutations would be an advantage as a screening tool to identify potentially-resistant strains and to track their movement. To complement DNA sequencing, we developed a padlock probe and rolling circle amplification (RCA-based method to detect a series of mutations in the C. albicans ERG11 gene using "reference" azole-resistant isolates with known mutations. The method was then used to estimate the frequency of ERG11 mutations and their type in 25 Australian clinical C. albicans isolates with reduced susceptibility to fluconazole and in 23 fluconazole-susceptible isolates. RCA results were compared DNA sequencing. Results The RCA assay correctly identified all ERG11 mutations in eight "reference" C. albicans isolates. When applied to 48 test strains, the RCA method showed 100% agreement with DNA sequencing where an ERG11 mutation-specific probe was used. Of 20 different missense mutations detected by sequencing in 24 of 25 (96% isolates with reduced fluconazole susceptibility, 16 were detected by RCA. Five missense mutations were detected by both methods in 18 of 23 (78% fluconazole-susceptible strains. DNA sequencing revealed that mutations in non-susceptible isolates were all due to homozygous nucleotide changes. With the exception of the mutations leading to amino acid substitution E266D, those in fluconazole-susceptible strains were heterozygous. Amino acid substitutions common to both sets of isolates were D116E, E266D, K128T, V437I and V488I. Substitutions unique to isolates with reduced fluconazole susceptibility were G464 S (n = 4 isolates, G448E (n = 3, G307S (n = 3, K143R (n = 3 and Y123H, S405F and R467K (each n = 1. DNA sequencing revealed a novel substitution, G450V, in one isolate. Conclusion The sensitive RCA

  8. Evaluation of Rapid Stain IDentification (RSID™ Reader System for Analysis and Documentation of RSID™ Tests

    Directory of Open Access Journals (Sweden)

    Pravatchai W. Boonlayangoor

    2013-08-01

    Full Text Available The ability to detect the presence of body fluids is a crucial first step in documenting and processing forensic evidence. The Rapid Stain IDentification (RSID™ tests for blood, saliva, semen and urine are lateral flow immunochromatographic strip tests specifically designed for forensic use. Like most lateral flow strips, the membrane components of the test are enclosed in a molded plastic cassette with a sample well and an observation window. No specialized equipment is required to use these tests or to score the results seen in the observation window; however, the utility of these tests can be enhanced if an electronic record of the test results can be obtained, preferably by a small hand-held device that could be used in the field under low light conditions. Such a device should also be able to “read” the lateral flow strips and accurately record the results of the test as either positive, i.e., the body fluid was detected, or negative, i.e., the body fluid was not detected. Here we describe the RSID™ Reader System—a ruggedized strip test reader unit that allows analysis and documentation of RSID™ lateral flow strip tests using pre-configured settings, and show that the RSID™ Reader can accurately and reproducibly report and record correct results from RSID™ blood, saliva, semen, and urine tests.

  9. Rapid identification of drug resistant Candida species causing recurrent vulvovaginal candidiasis.

    Science.gov (United States)

    Diba, Kambiz; Namaki, Atefeh; Ayatolahi, Haleh; Hanifian, Haleh

    2012-01-01

    Some yeast agents including Candida albicans, Candida tropicalis and Candida glabrata have a role in recurrent vulvovaginal candidiasis. We studied the frequency of both common and recurrent vulvovaginal candidiasis in symptomatic cases which were referred to Urmia Medical Sciences University related gynecology clinics using morphologic and molecular methods. The aim of this study was the identification of Candida species isolated from recurrent vulvovaginal candidiasis cases using a rapid and reliable molecular method. Vaginal swabs obtained from each case, were cultured on differential media including cornmeal agar and CHROM agar Candida. After 48 hours at 37℃, the cultures were studied for growth characteristics and color production respectively. All isolates were identified using the molecular method of PCR - restriction fragment length polymorphism. Among all clinical specimens, we detected 19 ( 16 % ) non fungal agents, 87 ( 82.1 % ) yeasts and 2 ( 1.9 % ) multiple infections. The yeast isolates identified morphologically included Candida albicans ( n = 62 ), Candida glabrata ( n = 9 ), Candida tropicalis ( n = 8 ), Candida parapsilosis ( n = 8 ) and Candida guilliermondii and Candida krusei ( n = 1 each ). We also obtained very similar results for Candida albicans, Candida glabrata and Candida tropicalis as the most common clinical isolates, by using PCR - Restriction Fragment Length Polymorphism. Use of two differential methods, morphologic and molecular, enabled us to identify most medically important Candida species which particularly cause recurrent vulvovaginal candidiasis.

  10. Method for rapid detection and identification of chaetomium and evaluation of resistance to peracetic acid.

    Science.gov (United States)

    Nakayama, Motokazu; Hosoya, Kouichi; Tomiyama, Daisuke; Tsugukuni, Takashi; Matsuzawa, Tetsuhiro; Imanishi, Yumi; Yaguchi, Takashi

    2013-06-01

    In the beverage industry, peracetic acid has been increasingly used as a disinfectant for the filling machinery and environment due to merits of leaving no residue, it is safe for humans, and its antiseptic effect against fungi and endospores of bacteria. Recently, Chaetomium globosum and Chaetomium funicola were reported resistant to peracetic acid; however, little is known concerning the detail of peracetic acid resistance. Therefore, we assessed the peracetic acid resistance of the species of Chaetomium and related genera under identical conditions and made a thorough observation of the microstructure of their ascospores by transmission electron microscopy. The results of analyses revealed that C. globosum and C. funicola showed the high resistance to peracetic acid (a 1-D antiseptic effect after 900 s and 3-D antiseptic effect after 900 s) and had thick cell walls of ascospores that can impede the action mechanism of peracetic acid. We also developed specific primers to detect the C. globosum clade and identify C. funicola by using PCR to amplify the β-tubulin gene. PCR with the primer sets designed for C. globosum (Chae 4F/4R) and C. funicola (Cfu 2F/2R) amplified PCR products specific for the C. globosum clade and C. funicola, respectively. PCR with these two primer sets did not detect other fungi involved in food spoilage and environmental contamination. This detection and identification method is rapid and simple, with extremely high specificity.

  11. Network Understanding of Herb Medicine via Rapid Identification of Ingredient-Target Interactions

    Science.gov (United States)

    Zhang, Hai-Ping; Pan, Jian-Bo; Zhang, Chi; Ji, Nan; Wang, Hao; Ji, Zhi-Liang

    2014-01-01

    Today, herb medicines have become the major source for discovery of novel agents in countermining diseases. However, many of them are largely under-explored in pharmacology due to the limitation of current experimental approaches. Therefore, we proposed a computational framework in this study for network understanding of herb pharmacology via rapid identification of putative ingredient-target interactions in human structural proteome level. A marketing anti-cancer herb medicine in China, Yadanzi (Brucea javanica), was chosen for mechanistic study. Total 7,119 ingredient-target interactions were identified for thirteen Yadanzi active ingredients. Among them, about 29.5% were estimated to have better binding affinity than their corresponding marketing drug-target interactions. Further Bioinformatics analyses suggest that simultaneous manipulation of multiple proteins in the MAPK signaling pathway and the phosphorylation process of anti-apoptosis may largely answer for Yadanzi against non-small cell lung cancers. In summary, our strategy provides an efficient however economic solution for systematic understanding of herbs' power.

  12. Identification of missense mutations in the Norrie disease gene associated with advanced retinopathy of prematurity.

    Science.gov (United States)

    Shastry, B S; Pendergast, S D; Hartzer, M K; Liu, X; Trese, M T

    1997-05-01

    Retinopathy of prematurity (ROP) is a retinal vascular disease occurring in infants with short gestational age and low birth weight and can lead to retinal detachment (ROP stages 4 and 5). X-linked familial exudative vitreoretinopathy is phenotypically similar to ROP and has been associated with mutations in the Norrie disease (ND) gene in some cases. To determine if similar mutations in the ND gene may play a role in the development of advanced ROP. Clinical examination and molecular genetic analysis were performed on 16 children, including 2 dizygotic and 1 monozygotic twin pairs, and their parents from 13 families. Sequencing of the amplified products revealed missense mutations (R121W and L108P) in the third exon of the ND gene in 4 patients. These mutations were not present in an unaffected premature twin, 2 children with regressed stage 3 ROP, the parents, or in 50 unrelated healthy control subjects. These findings suggest that mutations in the ND gene may play a role in the development of severe ROP in premature infants.

  13. Long-range PCR facilitates the identification of PMS2-specific mutations.

    Science.gov (United States)

    Clendenning, Mark; Hampel, Heather; LaJeunesse, Jennifer; Lindblom, Annika; Lockman, Jan; Nilbert, Mef; Senter, Leigha; Sotamaa, Kaisa; de la Chapelle, Albert

    2006-05-01

    Mutations within the DNA mismatch repair gene, "postmeiotic segregation increased 2" (PMS2), have been associated with a predisposition to hereditary nonpolyposis colorectal cancer (HNPCC; Lynch syndrome). The presence of a large family of highly homologous PMS2 pseudogenes has made previous attempts to sequence PMS2 very difficult. Here, we describe a novel method that utilizes long-range PCR as a way to preferentially amplify PMS2 and not the pseudogenes. A second, exon-specific, amplification from diluted long-range products enables us to obtain a clean sequence that shows no evidence of pseudogene contamination. This method has been used to screen a cohort of patients whose tumors were negative for the PMS2 protein by immunohistochemistry and had not shown any mutations within the MLH1 gene. Sequencing of the PMS2 gene from 30 colorectal and 11 endometrial cancer patients identified 10 novel sequence changes as well as 17 sequence changes that had previously been identified. In total, putative pathologic mutations were detected in 11 of the 41 families. Among these were five novel mutations, c.705+1G>T, c.736_741del6ins11, c.862_863del, c.1688G>T, and c.2007-1G>A. We conclude that PMS2 mutation detection in selected Lynch syndrome and Lynch syndrome-like patients is both feasible and desirable. Published 2006 Wiley-Liss, Inc.

  14. Identification of ALK germline mutation (3605delG) in pediatric anaplastic medulloblastoma.

    Science.gov (United States)

    Coco, Simona; De Mariano, Marilena; Valdora, Francesca; Servidei, Tiziana; Ridola, Vita; Andolfo, Immacolata; Oberthuer, André; Tonini, Gian Paolo; Longo, Luca

    2012-10-01

    The anaplastic lymphoma kinase (ALK) gene has been found either rearranged or mutated in several neoplasms such as anaplastic large-cell lymphoma, non-small-cell lung cancer, neuroblastoma and anaplastic thyroid cancer. Medulloblastoma (MB) is an embryonic pediatric cancer arising from nervous system, a tissue in which ALK is expressed during embryonic development. We performed an ALK mutation screening in 52 MBs and we found a novel heterozygous germline deletion of a single base in exon 23 (3605delG) in a case with marked anaplasia. This G deletion results in a frameshift mutation producing a premature stop codon in exon 25 of ALK tyrosine kinase domain. We also screened three human MB cell lines without finding any mutation of ALK gene. Quantitative expression analysis of 16 out of 52 samples showed overexpression of ALK mRNA in three MBs. In the present study, we report the first mutation of ALK found in MB. Moreover, a deletion of ALK gene producing a stop codon has not been detected in human tumors up to now. Further investigations are now required to elucidate whether the truncated form of ALK may have a role in signal transduction.

  15. Hereditary thrombophilia: identification of nonsense and missense mutations in the protein C gene

    International Nuclear Information System (INIS)

    Romeo, G.; Hassan, H.J.; Staempfli, S.

    1987-01-01

    The structure of the gene for protein C, an anticoagulant serine protease, was analyzed in 29 unrelated patients with hereditary thrombophilia and protein C deficiency. Gene deletion(s) or gross rearrangement(s) was not demonstrable by Southern blot hybridization to cDNA probes. However, two unrelated patients showed a variant restriction pattern after Pvu II or BamHi digestion, due to mutations in the last exon: analysis of their pedigrees, including three or seven heterozygotes, respectively, with ∼50% reduction of both enzymatic and antigen level, showed the abnormal restriction pattern in all heterozygous individuals, but not in normal relatives. Cloning of protein C gene and sequencing of the last exon allowed the authors to identify a nonsense and a missense mutation, respectively. In the first case, codon 306 (CGA, arginine) is mutated to an inframe stop codon, thus generating a new Pvu II recognition site. In the second case, a missense mutation in the BamHI palindrome (GGATCC → GCATCC) leads to substitution of a key amino acid (a tryptophan to cysteine substitution at position 402), invariantly conserved in eukaryotic serine proteases. These point mutations may explain the protein C-deficiency phenotype of heterozygotes in the two pedigrees

  16. Identification of a novel MYO7A mutation in Usher syndrome type 1.

    Science.gov (United States)

    Cheng, Ling; Yu, Hongsong; Jiang, Yan; He, Juan; Pu, Sisi; Li, Xin; Zhang, Li

    2018-01-05

    Usher syndrome (USH) is an autosomal recessive disease characterized by deafness and retinitis pigmentosa. In view of the high phenotypic and genetic heterogeneity in USH, performing genetic screening with traditional methods is impractical. In the present study, we carried out targeted next-generation sequencing (NGS) to uncover the underlying gene in an USH family (2 USH patients and 15 unaffected relatives). One hundred and thirty-five genes associated with inherited retinal degeneration were selected for deep exome sequencing. Subsequently, variant analysis, Sanger validation and segregation tests were utilized to identify the disease-causing mutations in this family. All affected individuals had a classic USH type I (USH1) phenotype which included deafness, vestibular dysfunction and retinitis pigmentosa. Targeted NGS and Sanger sequencing validation suggested that USH1 patients carried an unreported splice site mutation, c.5168+1G>A, as a compound heterozygous mutation with c.6070C>T (p.R2024X) in the MYO7A gene. A functional study revealed decreased expression of the MYO7A gene in the individuals carrying heterozygous mutations. In conclusion, targeted next-generation sequencing provided a comprehensive and efficient diagnosis for USH1. This study revealed the genetic defects in the MYO7A gene and expanded the spectrum of clinical phenotypes associated with USH1 mutations.

  17. Identification of Missense Mutation (I12T in the BSND Gene and Bioinformatics Analysis

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    Hina Iqbal

    2011-01-01

    Full Text Available Nonsyndromic hearing loss is a paradigm of genetic heterogeneity with 85 loci and 39 nuclear disease genes reported so far. Mutations of BSND have been shown to cause Bartter syndrome type IV, characterized by significant renal abnormalities and deafness and nonsyndromic nearing loss. We studied a Pakistani consanguineous family. Clinical examinations of affected individuals did not reveal the presence of any associated signs, which are hallmarks of the Bartter syndrome type IV. Linkage analysis identified an area of 18.36 Mb shared by all affected individuals between markers D1S2706 and D1S1596. A maximum two-point LOD score of 2.55 with markers D1S2700 and multipoint LOD score of 3.42 with marker D1S1661 were obtained. BSND mutation, that is, p.I12T, cosegregated in all extant members of our pedigree. BSND mutations can cause nonsyndromic hearing loss, and it is a second report for this mutation. The respected protein, that is, BSND, was first modeled, and then, the identified mutation was further analyzed by using different bioinformatics tools; finally, this protein and its mutant was docked with CLCNKB and REN, interactions of BSND, respectively.

  18. Unexpected identification of a recurrent mutation in the DLX3 gene causing amelogenesis imperfecta.

    Science.gov (United States)

    Kim, Y-J; Seymen, F; Koruyucu, M; Kasimoglu, Y; Gencay, K; Shin, T J; Hyun, H-K; Lee, Z H; Kim, J-W

    2016-05-01

    To identify the molecular genetic aetiology of a family with autosomal dominant amelogenesis imperfecta (AI). DNA samples were collected from a six-generation family, and the candidate gene approach was used to screen for the enamelin (ENAM) gene. Whole-exome sequencing and linkage analysis with SNP array data identified linked regions, and candidate gene screening was performed. Mutational analysis revealed a mutation (c.561_562delCT and p.Tyr188Glnfs*13) in the DLX3 gene. After finding a recurrent DLX3 mutation, the clinical phenotype of the family members was re-examined. The proband's mother had pulp elongation in the third molars. The proband had not hair phenotype, but her cousin had curly hair at birth. In this study, we identified a recurrent 2-bp deletional DLX3 mutation in a new family. The clinical phenotype was the mildest one associated with the DLX3 mutations. These results will advance the understanding of the functional role of DLX3 in developmental processes. © 2016 The Authors. Oral Diseases Published by John Wiley & Sons Ltd.

  19. Identification of a Novel Homozygous Nonsense Mutation Confirms the Implication of GNAT1 in Rod-Cone Dystrophy.

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    Cécile Méjécase

    Full Text Available GNAT1, encoding the transducin subunit Gα, is an important element of the phototransduction cascade. Mutations in this gene have been associated with autosomal dominant and autosomal recessive congenital stationary night blindness. Recently, a homozygous truncating GNAT1 mutation was identified in a patient with late-onset rod-cone dystrophy. After exclusion of mutations in genes underlying progressive inherited retinal disorders, by targeted next generation sequencing, a 32 year-old male sporadic case with severe rod-cone dystrophy and his unaffected parents were investigated by whole exome sequencing. This led to the identification of a homozygous nonsense variant, c.963C>A p.(Cys321* in GNAT1, which was confirmed by Sanger sequencing. The mother was heterozygous for this variant whereas the variant was absent in the father. c.963C>A p.(Cys321* is predicted to produce a shorter protein that lacks critical sites for the phototransduction cascade. Our work confirms that the phenotype and the mode of inheritance associated with GNAT1 variants can vary from autosomal dominant, autosomal recessive congenital stationary night blindness to autosomal recessive rod-cone dystrophy.

  20. Identification of Novel Mutations in Spatacsin and Apolipoprotein B Genes in a Patient with Spastic Paraplegia and Hypobetalipoproteinemia

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    Leema Reddy Peddareddygari

    2015-01-01

    Full Text Available Complicated hereditary spastic paraplegia (HSP presents with complex neurological and nonneurological manifestations. We report a patient with autosomal recessive (AR HSP in whom laboratory investigations revealed hypobetalipoproteinemia raising the possibility of a shared pathophysiology of these clinical features. A lipid profile of his parents disclosed a normal maternal lipid profile. However, the paternal lipid profile was similar to that of the patient suggesting autosomal dominant transmission of this trait. Whole exome sequence analysis was performed and novel mutations were detected in both the SPG11 and the APOB genes. Genetic testing of the parents showed that both APOB variants were inherited from the father while the SPG11 variants were inherited one from each parent. Our results indicate that, in this patient, the hypobetalipoproteinemia and spastic paraplegia are unrelated resulting from mutations in two independent genes. This clinical study provides support for the use of whole exome sequencing as a diagnostic tool for identification of mutations in conditions with complex presentations.

  1. Identification and Genetic Analysis of a Factor IX Gene Intron 3 Mutation in a Hemophilia B Pedigree in China

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    Dong Hua Cao

    2014-09-01

    Full Text Available OBJECTIVE: Hemophilia B is caused by coagulation defects in the factor IX gene located in Xq27.1 on the X chromosome. A wide range of mutations, showing extensive molecular heterogeneity, have been described in hemophilia B patients. Our study was aimed at genetic analysis and prenatal diagnosis of hemophilia B in order to further elucidate the pathogenesis of the hemophilia B pedigree in China. METHODS: Polymerase chain reaction amplification and direct sequencing of all the coding regions was conducted in hemophilia B patients and carriers. Prenatal diagnosis of the proband was conducted at 20 weeks. RESULTS: We identified the novel point mutation 10.389 A>G, located upstream of the intron 3 acceptor site in hemophilia B patients. The fetus of the proband’s cousin was identified as a carrier. CONCLUSION: Our identification of a novel mutation in the F9 gene associated with hemophilia B provides novel insight into the pathogenesis of this genetically inherited disorder and also represents the basis of prenatal diagnosis.

  2. High-risk Long QT Syndrome Mutations in the Kv7.1 (KCNQ1) Pore Disrupt the Molecular Basis for Rapid K+ Permeation

    Science.gov (United States)

    Burgess, Don E.; Bartos, Daniel C.; Reloj, Allison R.; Campbell, Kenneth S.; Johnson, Jonathan N.; Tester, David J.; Ackerman, Michael J.; Fressart, Véronique; Denjoy, Isabelle; Guicheney, Pascale; Moss, Arthur J.; Ohno, Seiko; Horie, Minoru; Delisle, Brian P.

    2012-01-01

    Type 1 long QT syndrome (LQT1) syndrome is caused by loss-of-function mutations in the KCNQ1, which encodes the K+ channel (Kv7.1) that underlies the slowly activating delayed rectifier K+ current in the heart. Intragenic risk stratification suggests LQT1 mutations that disrupt conserved amino acid residues in the pore are an independent risk factor for LQT1-related cardiac events. The purpose of this study is to determine possible molecular mechanisms that underlie the loss-of-function for these high-risk mutations. Extensive genotype-phenotype analyses of LQT1 patients showed that T322M-, T322A-, or G325R-Kv7.1 confer a high risk for LQT1-related cardiac events. Heterologous expression of these mutations with KCNE1 revealed they generated non-functional channels and caused dominant negative suppression of WT-Kv7.1 current. Molecular dynamic simulations (MDS) of analogous mutations in KcsA (T85M-, T85A-, and G88R-KcsA) demonstrated that they disrupted the symmetrical distribution of the carbonyl oxygen atoms in the selectivity filter, which upset the balance between the strong attractive and K+-K+ repulsive forces required for rapid K+ permeation. We conclude high-risk LQT1 mutations in the pore likely disrupt the architectural and physical properties of the K+ channel selectivity filter. PMID:23092362

  3. High-risk long QT syndrome mutations in the Kv7.1 (KCNQ1) pore disrupt the molecular basis for rapid K(+) permeation.

    Science.gov (United States)

    Burgess, Don E; Bartos, Daniel C; Reloj, Allison R; Campbell, Kenneth S; Johnson, Jonathan N; Tester, David J; Ackerman, Michael J; Fressart, Véronique; Denjoy, Isabelle; Guicheney, Pascale; Moss, Arthur J; Ohno, Seiko; Horie, Minoru; Delisle, Brian P

    2012-11-13

    Type 1 long QT syndrome (LQT1) is caused by loss-of-function mutations in the KCNQ1 gene, which encodes the K(+) channel (Kv7.1) that underlies the slowly activating delayed rectifier K(+) current in the heart. Intragenic risk stratification suggests LQT1 mutations that disrupt conserved amino acid residues in the pore are an independent risk factor for LQT1-related cardiac events. The purpose of this study is to determine possible molecular mechanisms that underlie the loss of function for these high-risk mutations. Extensive genotype-phenotype analyses of LQT1 patients showed that T322M-, T322A-, or G325R-Kv7.1 confers a high risk for LQT1-related cardiac events. Heterologous expression of these mutations with KCNE1 revealed they generated nonfunctional channels and caused dominant negative suppression of WT-Kv7.1 current. Molecular dynamics simulations of analogous mutations in KcsA (T85M-, T85A-, and G88R-KcsA) demonstrated that they disrupted the symmetrical distribution of the carbonyl oxygen atoms in the selectivity filter, which upset the balance between the strong attractive and K(+)-K(+) repulsive forces required for rapid K(+) permeation. We conclude high-risk LQT1 mutations in the pore likely disrupt the architectural and physical properties of the K(+) channel selectivity filter.

  4. Next Generation Sequencing approach to molecular diagnosis of Duchenne muscular dystrophy; identification of a novel mutation.

    Science.gov (United States)

    Ebrahimzadeh-Vesal, Reza; Teymoori, Atieh; Azimi-Nezhad, Mohsen; Hosseini, Forough Sadat

    2018-02-20

    Duchenne Muscular Dystrophy (DMD; MIM 310200) is one of the most common and severe type of hereditary muscular dystrophies. The disease is caused by mutations in the dystrophin gene. The dystrophin gene is associated with X-linked recessive Duchenne and Becker muscular dystrophy. This disease occurs almost exclusively in males. The clinical symptoms of muscle weakness usually begin at childhood. The main symptoms of this disorder are gradually muscular weakness. The affected patients have inability to standing up and walking. Death is usually due to respiratory infection or cardiomyopathy. In this article, we have reported the discovery of a new nonsense mutation that creates abnormal stop codon in the dystrophin gene. This mutation was detected using Next Generation Sequencing (NGS) technique. The subject was a 17-year-old male with muscular dystrophy that who was suspected of having DMD. He was referred to Hakim medical genetics center of Neyshabur, IRAN. Copyright © 2017. Published by Elsevier B.V.

  5. Identification of weak points prone for mutation in ferredoxin of Trichomonas vaginalis

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    Wiwanitkit V

    2008-01-01

    Full Text Available Trichomonas vaginalis , the causative agent for human trichomoniasis, is a problematic sexually transmitted disease mainly in women. At present, metronidazole-resistant trichomoniasis is an infrequent but challenging problem with no universally successful treatment. Genetic mutation is believed to be an important factor leading to increasing drug resistance. Understanding the mutation status will help to design accurate strategies of therapy against mutant strains of T. vaginalis . The author performed a bioinformatic analysis to determine positions that tend to comply peptide motifs in the amino acid sequence of ferridoxin of T. vaginalis . Based on this study, the weak linkages in the studied protein can be identified and can be useful information for prediction of possible new mutations that can lead to drug resistance. In addition, the results from this study can be good information for further research on the diagnosis for mutants and new effective drug development.

  6. Identification of a Novel HADHB Gene Mutation in an Iranian Patient with Mitochondrial Trifunctional Protein Deficiency.

    Science.gov (United States)

    Shahrokhi, Mahdiyeh; Shafiei, Mohammad; Galehdari, Hamid; Shariati, Gholamreza

    2017-01-01

    Mitochondrial trifunctional protein (MTP) is a hetero-octamer composed of eight parts (subunits): four α-subunits containing LCEH (long-chain 2,3-enoyl-CoA  hydratase) and LCHAD (long-chain 3-hydroxyacyl CoA dehydrogenase) activity, and four β-subunits that possess LCKT (long-chain  3-ketoacyl-CoA thiolase) activity which catalyzes three out of four steps in β-oxidation spiral of long-chain fatty acid. Its deficiency is an autosomal recessive disorder that causes a clinical spectrum of diseases. A blood spot was collected from the patient's original newborn screening card with parental informed consent. A newborn screening test and quantity plasma acylcarnitine profile analysis by MS/MS were performed. After isolation of DNA and Amplification of all exons of the HADHA and HADHB, directly Sequence analyses of all exons and the flanking introns both of genes were performed. Here, we report a novel mutation in a patient with MTP deficiency diagnosed with newborn screening test and quantity plasma acylcarnitine profile analysis by MS/MS and then confirmed by enzyme analysis in cultured fibroblasts and direct sequencing of the HADHA and HADHB genes. Molecular analysis of causative genes showed a missense mutation (p.Q385P) c.1154A > C in exon 14 of HADHB gene. Since this mutation was not found in 50 normal control cases; so it was concluded that c.1154A > C mutation was a causative mutation. Phenotype analysis of this mutation predicted pathogenesis which reduces the stability of the MTP protein complex.

  7. Identification of eight novel coagulation factor XIII subunit A mutations: implied consequences for structure and function.

    Science.gov (United States)

    Ivaskevicius, Vytautas; Biswas, Arijit; Bevans, Carville; Schroeder, Verena; Kohler, Hans Peter; Rott, Hannelore; Halimeh, Susan; Petrides, Petro E; Lenk, Harald; Krause, Manuele; Miterski, Bruno; Harbrecht, Ursula; Oldenburg, Johannes

    2010-06-01

    Severe hereditary coagulation factor XIII deficiency is a rare homozygous bleeding disorder affecting one person in every two million individuals. In contrast, heterozygous factor XIII deficiency is more common, but usually not associated with severe hemorrhage such as intracranial bleeding or hemarthrosis. In most cases, the disease is caused by F13A gene mutations. Causative mutations associated with the F13B gene are rarer. We analyzed ten index patients and three relatives for factor XIII activity using a photometric assay and sequenced their F13A and F13B genes. Additionally, structural analysis of the wild-type protein structure from a previously reported X-ray crystallographic model identified potential structural and functional effects of the missense mutations. All individuals except one were heterozygous for factor XIIIA mutations (average factor XIII activity 51%), while the remaining homozygous individual was found to have severe factor XIII deficiency (<5% of normal factor XIII activity). Eight of the 12 heterozygous patients exhibited a bleeding tendency upon provocation. The identified missense (Pro289Arg, Arg611His, Asp668Gly) and nonsense (Gly390X, Trp664X) mutations are causative for factor XIII deficiency. A Gly592Ser variant identified in three unrelated index patients, as well as in 200 healthy controls (minor allele frequency 0.005), and two further Tyr167Cys and Arg540Gln variants, represent possible candidates for rare F13A gene polymorphisms since they apparently do not have a significant influence on the structure of the factor XIIIA protein. Future in vitro expression studies of the factor XIII mutations are required to confirm their pathological mechanisms.

  8. A rapid and effective method for screening, sequencing and reporter verification of engineered frameshift mutations in zebrafish

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    Sergey V. Prykhozhij

    2017-06-01

    Full Text Available Clustered regularly interspaced palindromic repeats (CRISPR/Cas-based adaptive immunity against pathogens in bacteria has been adapted for genome editing and applied in zebrafish (Danio rerio to generate frameshift mutations in protein-coding genes. Although there are methods to detect, quantify and sequence CRISPR/Cas9-induced mutations, identifying mutations in F1 heterozygous fish remains challenging. Additionally, sequencing a mutation and assuming that it causes a frameshift does not prove causality because of possible alternative translation start sites and potential effects of mutations on splicing. This problem is compounded by the relatively few antibodies available for zebrafish proteins, limiting validation at the protein level. To address these issues, we developed a detailed protocol to screen F1 mutation carriers, and clone and sequence identified mutations. In order to verify that mutations actually cause frameshifts, we created a fluorescent reporter system that can detect frameshift efficiency based on the cloning of wild-type and mutant cDNA fragments and their expression levels. As proof of principle, we applied this strategy to three CRISPR/Cas9-induced mutations in pycr1a, chd7 and hace1 genes. An insertion of seven nucleotides in pycr1a resulted in the first reported observation of exon skipping by CRISPR/Cas9-induced mutations in zebrafish. However, of these three mutant genes, the fluorescent reporter revealed effective frameshifting exclusively in the case of a two-nucleotide deletion in chd7, suggesting activity of alternative translation sites in the other two mutants even though pycr1a exon-skipping deletion is likely to be deleterious. This article provides a protocol for characterizing frameshift mutations in zebrafish, and highlights the importance of checking mutations at the mRNA level and verifying their effects on translation by fluorescent reporters when antibody detection of protein loss is not possible.

  9. Identification of a novel mutation in HPS6 in a patient with hemophilia B and oculocutaneous albinism.

    Science.gov (United States)

    O'Brien, Kevin J; Lozier, Jay; Cullinane, Andrew R; Osorio, Brigitte; Nghiem, Khanh; Speransky, Vladislav; Zein, Wadih M; Mullikin, James C; Neff, Anne T; Simon, Karen L; Malicdan, May Christine V; Gahl, William A; Young, Lisa R; Gochuico, Bernadette R

    2016-11-01

    Hemophilia B, an X-linked disease, manifests with recurrent soft tissue bleeding episodes. Hermansky-Pudlak syndrome, a rare autosomal recessive disorder, is characterized by oculocutaneous albinism and an increased tendency to bleed due to a platelet storage pool defect. We report a novel mutation in HPS6 in a Caucasian man with hemophilia B and oculocutaneous albinism. The patient was diagnosed with hemophilia B at age 4months due to recurrent soft tissue bleeding episodes, and he was also diagnosed with Hermansky-Pudlak syndrome at 32years of age due to unexplained oculocutaneous albinism. His factor IX level was markedly reduced at 13%; whole exome and Sanger sequencing showed the Durham mutation in F9 (NM_000133.3). The diagnosis of Hermansky-Pudlak syndrome subtype 6 was established by demonstrating absence of platelet delta granules on whole mount electron microscopy, an abnormal secondary wave in platelet aggregation studies, and a novel homozygous c.1114 C>T (p.Arg372*) mutation in HPS6 (NM_024747.5) on exome analysis and Sanger sequencing. Clinical phenotyping revealed no evidence of recurrent or unusual infections, interstitial lung disease or pulmonary fibrosis, or neurological disorders. The patient was treated with fresh frozen plasma, recombinant factor IX, and aminocaproic acid. Treatment with desmopressin was added to his regimen after he was diagnosed with Hermansky-Pudlak syndrome. Treatment of bleeding episodes results in effective hemostasis, and the patient has not required platelet or blood product transfusions. This report highlights the need to consider Hermansky-Pudlak syndrome as an etiology of oculocutaneous albinism even in patients with known hematologic disorders associated with bleeding. Identification of a novel mutation in HPS6 in an individual with hemophilia B shows that, although quite rare, patients may be diagnosed with two independent inherited bleeding disorders. No evidence of lung disease was found in this adult patient

  10. Identification of the second mutation of BADH2 gene derived from rice mutant lines induced by gamma rays

    International Nuclear Information System (INIS)

    I Ishak

    2016-01-01

    The BADH2 gene acts as suppressor of 2-acetyl-1-pyrolline (2AP) biosynthesis in plants. 2AP is the volatile compound which provides fragrance in rice. Biosynthesis of 2AP occurs when BADH2 loses its function as suppressor gene. Aromatic rice cultivars naturally incur mutation of BADH2 gene at 8 bp. In this experiment, aromatic mutant rice lines derived from irradiation of Sintanur cultivar by gamma rays with dose of 100 Gy were studied in molecular level. These mutant lines were characterized at the M10 plantgeneration under the assumption that genetically these aromatic mutant rice lines were homozygotic. Several primers related to aroma in rice have been used for polymerase chain reaction (PCR) in a thermal cycler instrument. Gel electrophoreses were carried out using 1.5% agarose in TAE buffer. DNA fragments at 254 bp and 355 bp (base pair) were taken and amplified by primer for nucleotide sequencing of these fragments. Molecular identification and characterization after electrophoresis showed that the mutant line from AR1020 can be differentiated from AR.1080 at 254 bp. Nucleotide sequence data from of these DNA fragments showed that point mutations (deletions and substitutions) occurred at the BADH2 gene in exon 7; those are called second mutation and were caused by gamma rays effects. The Sintanur variety was used as check cultivar and its DNA sequence was compared to that of the AR.1020 mutant line. The results from both DNA sequences (from cv. Sintanur and AR.1020) derived from fragments at 254 bp show that point mutations occurred within exon 7 and earlier stop codon occurred in the AR.1020 mutant rice line. Further, the use of EA primer in PCR resulted in detection of deletion and substitution of nucleotides in the AR.1020 mutant line. (author)

  11. Vitek 2 ANC card versus BBL Crystal Anaerobe and RapID ANA II for identification of clinical anaerobic bacteria.

    Science.gov (United States)

    Blairon, Laurent; Maza, Mengi L; Wybo, Ingrid; Piérard, Denis; Dediste, Anne; Vandenberg, Olivier

    2010-08-01

    The Vitek 2 Anaerobe and Corynebacterium Identification Card (ANC) was recently evaluated in a multicentre study. In the present work, this system was compared with the BBL Crystal Anaerobe and RapID ANA II panels. These kits were tested using 196 strains of anaerobes that had been previously identified by gas-liquid chromatography. Identification to the species or to the genus level was 75.0%, 81.1% and 70.9% for Crystal, RapID and Vitek, respectively. Vitek ANC failed to provide any identification in 20.4% of the strains, but it had fewer misidentifications than RapID. The confidence factors provided on the results report of each kit were not always correlated with a lower risk of major errors, with the exception of Vitek 2 in which a confidence factor higher than 0.86 excluded the risk of misidentification in more than 87% of isolates. The lower rate of identification by the Vitek and Crystal panels is mostly due the lower ability of these systems to identify the Clostridia. Overall, the three panels are comparable but need improvement to a better accuracy. Copyright (c) 2010 Elsevier Ltd. All rights reserved.

  12. Comparison of four methods for rapid identification of Staphylococcus aureus directly from BACTEC 9240 blood culture system

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    N S Ozen

    2011-01-01

    Full Text Available Purpose: Differentiation of Staphylococcus aureus (S. aureus from coagulase-negative staphylococci is very important in blood stream infections. Identification of S. aureus and coagulase-negative staphylococci (CoNS from blood cultures takes generally 18-24 h after positive signaling on continuously monitored automated blood culture system. In this study, we evaluated the performance of tube coagulase test (TCT, slide agglutination test (Dry Spot Staphytect Plus, conventional polymerase chain reaction (PCR and LightCycler Staphylococcus MGrade kit directly from blood culture bottles to achieve rapid identification of S. aureus by using the BACTEC 9240 blood culture system. Materials and Methods: A total of 129 BACTEC 9240 bottles growing gram-positive cocci suggesting Staphylococci were tested directly from blood culture broths (BCBs with TCT, Dry Spot Staphytect Plus, conventional PCR and LightCycler Staphylococcus MGrade kit for rapid identification of S. aureus. Results: The sensitivities of the tests were 99, 68, 99 and 100%, respectively. Conclusion: Our results suggested that 2 h TCT was found to be simple and inexpensive method for the rapid identification of S. aureus directly from positive blood cultures.

  13. Comparison of four methods for rapid identification of Staphylococcus aureus directly from BACTEC 9240 blood culture system.

    Science.gov (United States)

    Ozen, N S; Ogunc, D; Mutlu, D; Ongut, G; Baysan, B O; Gunseren, F

    2011-01-01

    Differentiation of Staphylococcus aureus (S. aureus) from coagulase-negative staphylococci is very important in blood stream infections. Identification of S. aureus and coagulase-negative staphylococci (CoNS) from blood cultures takes generally 18-24 h after positive signaling on continuously monitored automated blood culture system. In this study, we evaluated the performance of tube coagulase test (TCT), slide agglutination test (Dry Spot Staphytect Plus), conventional polymerase chain reaction (PCR) and LightCycler Staphylococcus MGrade kit directly from blood culture bottles to achieve rapid identification of S. aureus by using the BACTEC 9240 blood culture system. A total of 129 BACTEC 9240 bottles growing gram-positive cocci suggesting Staphylococci were tested directly from blood culture broths (BCBs) with TCT, Dry Spot Staphytect Plus, conventional PCR and LightCycler Staphylococcus MGrade kit for rapid identification of S. aureus. The sensitivities of the tests were 99, 68, 99 and 100%, respectively. Our results suggested that 2 h TCT was found to be simple and inexpensive method for the rapid identification of S. aureus directly from positive blood cultures.

  14. Identification of a novel BRCA1 nucleotide 4803delCC/c.4684delCC mutation and a nucleotide 249T>A/c.130T>A (p.Cys44Ser) mutation in two Greenlandic Inuit families

    DEFF Research Database (Denmark)

    Hansen, Thomas van Overeem; Jønson, Lars; Albrechtsen, Anders

    2010-01-01

    Germ-line mutations in the tumour suppressor proteins BRCA1 and BRCA2 predispose to breast and ovarian cancer. We have recently identified a Greenlandic Inuit BRCA1 nucleotide 234T>G/c.115T>G (p.Cys39Gly) founder mutation, which at that time was the only disease-causing BRCA1/BRCA2 mutation...... identified in this population. Here, we describe the identification of a novel disease-causing BRCA1 nucleotide 4803delCC/c.4684delCC mutation in a Greenlandic Inuit with ovarian cancer. The mutation introduces a frameshift and a premature stop at codon 1572. We have also identified a BRCA1 nucleotide 249T......>A/c.130T>A (p.Cys44Ser) mutation in another Greenlandic individual with ovarian cancer. This patient share a 1-2 Mb genomic fragment, containing the BRCA1 gene, with four Danish families harbouring the same mutation, suggesting that the 249T>A/c.130T>A (p.Cys44Ser) mutation originates from a Danish...

  15. Identification of a KCNE2 gain-of-function mutation in patients with familial atrial fibrillation

    DEFF Research Database (Denmark)

    Yang, Yiqing; Xia, Min; Jin, Qingfeng

    2004-01-01

    Atrial fibrillation (AF) is the most common cardiac arrhythmia encountered in clinical practice. We first reported an S140G mutation of KCNQ1, an alpha subunit of potassium channels, in one Chinese kindred with AF. However, the molecular defects and cellular mechanisms in most patients with AF...

  16. Identification of the GDF9 mutation in two sheep breeds by using ...

    African Journals Online (AJOL)

    A genetic mutation with major effects on the litter size in sheep was recently identified in the growth differentiation factor (GDF9) gene of the TGF-B super family (transforming growth factor). GDF9 gene has been localized to chromosome 5 in sheep. In order to evaluate the GDF9 gene polymorphism, blood samples were ...

  17. Identification of a novel FBN1 gene mutation in a large Pakistani family with Marfan syndrome

    NARCIS (Netherlands)

    Micheal, S.; Khan, M.I.; Akhtar, F.; Weiss, M.M.; Islam, F.; Ali, M.; Qamar, R.; Maugeri, A.; Hollander, A.I. den

    2012-01-01

    PURPOSE: To describe a novel mutation in the fibrillin-1 (FBN1) gene in a large Pakistani family with autosomal dominant Marfan syndrome (MFS). METHODS: Blood samples were collected of 11 family members affected with Marfan syndrome, and DNA was isolated by phenol-extraction. The coding exons of

  18. Brody disease: insights into biochemical features of SERCA1 and identification of a novel mutation.

    NARCIS (Netherlands)

    Vattemi, G.; Gualandi, F.; Oosterhof, A.; Marini, M.; Tonin, P.; Rimessi, P.; Neri, M.; Guglielmi, V.; Russignan, A.; Poli, C.; Kuppevelt, A.H.M.S.M. van; Ferlini, A.; Tomelleri, G.

    2010-01-01

    Brody disease is an inherited disorder of skeletal muscle function characterized by increasing impairment of relaxation during exercise. The autosomal recessive form can be caused by mutations in the ATP2A1 gene, which encodes for the sarcoplasmic/endoplasmic reticulum Ca-ATPase 1 (SERCA1) protein.

  19. Identification of TBX5 mutations in a series of 94 patients with Tetralogy of Fallot

    NARCIS (Netherlands)

    Baban, Anwar; Postma, Alex Vincent; Marini, Monica; Trocchio, Gianluca; Santilli, Antonella; Pelegrini, Monica; Sirleto, Pietro; Lerone, Margherita; Albanese, Sonia Bernadette; Barnett, Phil; Boogerd, Cornelis Job; Dallapiccola, Bruno; Digilio, Maria Cristina; Ravazzolo, Roberto; Pongiglione, Giacomo

    2014-01-01

    Tetralogy of Fallot (TOF) (OMIM #187500) is the most frequent conotruncal congenital heart defect (CHD) with a range of intra- and extracardiac phenotypes. TBX5 is a transcription factor with well-defined roles in heart and forelimb development, and mutations in TBX5 are associated with Holt-Oram

  20. Rapid identification of Pterocarpus santalinus and Dalbergia louvelii by FTIR and 2D correlation IR spectroscopy

    Science.gov (United States)

    Zhang, Fang-Da; Xu, Chang-Hua; Li, Ming-Yu; Huang, An-Min; Sun, Su-Qin

    2014-07-01

    Since Pterocarpus santalinus and Dalbergia louvelii, which are of precious Rosewood, are very similar in their appearance and anatomy characteristics, cheaper Hongmu D. louvelii is often illegally used to impersonate valuable P. santalinus, especially in Chinese furniture manufacture. In order to develop a rapid and effective method for easy confused wood furniture differentiation, we applied tri-step identification method, i.e., conventional infrared spectroscopy (FT-IR), second derivative infrared (SD-IR) spectroscopy and two-dimensional correlation infrared (2DCOS-IR) spectroscopy to investigate P. santalinus and D. louvelii furniture. According to FT-IR and SD-IR spectra, it has been found two unconditional stable difference at 848 cm-1 and 700 cm-1 and relative stable differences at 1735 cm-1, 1623 cm-1, 1614 cm-1, 1602 cm-1, 1509 cm-1, 1456 cm-1, 1200 cm-1, 1158 cm-1, 1055 cm-1, 1034 cm-1 and 895 cm-1 between D. louvelii and P. santalinus IR spectra. The stable discrepancy indicates that the category of extractives is different between the two species. Besides, the relative stable differences imply that the content of holocellulose in P. santalinus is more than that of D. louvelii, whereas the quantity of extractives in D. louvelii is higher. Furthermore, evident differences have been observed in their 2DCOS-IR spectra of 1550-1415 cm-1 and 1325-1030 cm-1. P. santalinus has two strong auto-peaks at 1459 cm-1 and 1467 cm-1, three mid-strong auto-peaks at 1518 cm-1, 1089 cm-1 and 1100 cm-1 and five weak auto-peaks at 1432 cm-1, 1437 cm-1, 1046 cm-1, 1056 cm-1 and 1307 cm-1 while D. louvelii has four strong auto-peaks at 1465 cm-1, 1523 cm-1, 1084 cm-1 and 1100 cm-1, four mid-strong auto-peaks at 1430 cm-1, 1499 cm-1, 1505 cm-1 and 1056 cm-1 and two auto-peaks at 1540 cm-1 and 1284 cm-1. This study has proved that FT-IR integrated with 2DCOS-IR could be applicable for precious wood furniture authentication in a direct, rapid and holistic manner.

  1. Mutations Phe785Leu and Thr618Met in Na+, K+-ATPase, Associated with Familial Rapid-Onset Dystonia Parkinsonism, Interfere with Na+ Interaction by Distinct Mechanisms

    DEFF Research Database (Denmark)

    Schack, Vivien Rodacker; Toustrup-Jensen, Mads Schak; Vilsen, Bente

    The Na+, K+-ATPase plays key roles in brain function. Recently, missense mutations in the Na+, K+-ATPase were found associated with familial rapid-onset dystonia parkinsonism (FRDP). Here, we have characterized the functional consequences of FRDP mutations Phe785Leu and Thr618Met. Both mutations...... lead to functionally altered, but active, Na+, K+-pumps that display reduced apparent affinity for cytoplasmic Na+, but the underlying mechanism differs between the mutants. In Phe785Leu, the interaction of the E1 form with Na+ is defective, and the E1-E2 equilibrium is not displaced. In Thr618Met......, the Na+ affinity is reduced because of displacement of the conformational equilibrium in favor of the K+-occluded E2(K2) form. In both mutants, K+ interaction at the external activating sites of the E2P phosphoenzyme is normal. The change of cellular Na+ homeostasis is likely a major factor contributing...

  2. Mutations Phe785Leu and Thr618Met in Na+, K+-ATPase, Associated with Familial Rapid-Onset Dystonia Parkinsonism, Interfere with Na+ Interaction by Distinct Mechanisms

    DEFF Research Database (Denmark)

    Schack, Vivien Rodacker; Toustrup-Jensen, Mads Schak; Vilsen, Bente

    The Na+, K+-ATPase plays key roles in brain function. Recently, missense mutations in the Na+, K+-ATPase were found associated with familial rapid-onset dystonia parkinsonism (FRDP). We have characterized the functional consequences of FRDP mutations Phe785Leu and Thr618Met. Both mutations lead...... to functionally altered, but active, Na+, K+-pumps that display reduced apparent affinity for cytoplasmic Na+, but the underlying mechanism differs between the mutants. In Phe785Leu, the interaction of the E1 form with Na+ is defective, and the E1-E2 equilibrium is not displaced. In Thr618Met, the Na+ affinity...... is reduced because of displacement of the conformational equilibrium in favor of the K+-occluded E2(K2) form. In both mutants, K+ interaction at the external activating sites of the E2P phosphoenzyme is normal. The change of cellular Na+ homeostasis is likely a major factor contributing to the development...

  3. A set of lacZ mutations in Escherichia coli that allow rapid detection of each of the six base substitutions

    International Nuclear Information System (INIS)

    Cupples, C.G.; Miller, J.H.

    1989-01-01

    We describe the construction of six strains of Escherichia coli with different mutations at the same coding position in the lacZ gene, which specifies the active site glutamic acid residue at position 461 in beta'-galactosidase. Each strain is Lac- and reverts to Lac+ only by restoring the glutamic acid codon. The strains have been designed so that each reverts via one of the six base substitutions. The set of strains allows detection of each transition and transversion simply by monitoring the Lac- to Lac+ frequency, as demonstrated here with characterized mutagens and mutator alleles. These strains are useful for rapidly determining the mutagenic specificity of mutagens at a single site, for detecting low levels of stimulation of certain base substitutions, for monitoring specific base changes in response to various experimental conditions or strain backgrounds, and for isolating new mutator strains

  4. Identification of novel splice site mutation IVS9 + 1(G > A) and novel complex allele G355R/R359X in Type 1 Gaucher patients heterozygous for mutation N370S.

    Science.gov (United States)

    Hoitsema, Kourtnee; Amato, Dominick; Khan, Aneal; Sirrs, Sandra; Choy, Francis Y M

    2016-09-01

    Gaucher disease is an autosomal recessive lysosomal storage disorder resulting from deficient glucocerebrosidase activity. More than 350 mutations that cause Gaucher disease have been described to date. Novel mutations can potentially provide insight into the glucocerebrosidase structure-function relationship and biochemical basis of the disease. Here, we report the identification of two novel mutations in two unrelated patients with type I (non-neuronopathic) Gaucher disease: 1) a splice site mutation IVS9 + 1G > A; and (2) a complex allele (cis) G355R/R359X. Both patients have a common N370S mutation in the other allele. The splice site mutation results from an intronic base substitution (G to A, c.1328 + 1, g.5005) at the donor splice site of exon and intron 9. The complex allele results from two point mutations in exon 8 of glucocerebrosidase (G to C at c.1180, g.4396, and T to C at c. 1192, g.4408) substituting glycine by arginine (G355R) and arginine by a premature termination (R359X), respectively. In order to demonstrate that G355R/R359X are in cis arrangement, PCR-amplified glucocerebrosidase exon 8 genomic DNA from the patient was cloned into the vector pJET1.2 in Escherichia coli TOP10® strain. Out of the 15 clones that were sequence analyzed, 10 contained the normal allele sequence and 5 contained the complex allele G355R/R359X sequence showing both mutations in cis arrangement. Restriction fragment length polymorphism analysis using Hph1 restriction endonuclease digest was established for the IVS9 + 1G > A mutation for confirmation and efficient identification of this mutation in future patients. Past literature suggests that mutations affecting splicing patterns of the glucocerebrosidase transcript as well as mutations in Gaucher complex alleles are detrimental to enzyme activity. However, compound heterozygosity with N370S, a mild mutation, will lead to a mild phenotype. The cases reported here support these past findings.

  5. Investigation into spore coat properties for the rapid identification of endospores in marine sediments

    Science.gov (United States)

    Rattray, J. E.; Chakraborty, A.; Bernard, B. B.; Brooks, J.; Hubert, C. R.

    2017-12-01

    Understanding the sediment biogeography of dormant marine thermophilic bacterial endospores (thermospores) has the potential to assist locating and characterising working petroleum systems. The presence of thermospores in cold ocean environments suggests that distribution occurs via hydrocarbon seepage from thermally active reservoirs. Low abundance and endospore coat physiology mean nucleic acid based techniques have limited success for in situ detection of thermospores. Alternative rapid analytical methods are needed so we investigated using the Schaeffer-Fulton (malachite green and safranin) and DAPI (4',6-diamidino-2-phenylindole) staining techniques on thermospores from cultures and marine sediments. Sediment samples from 111 locations in the Eastern Gulf of Mexico (100 to 3300 m water depth; 6 to 600 km apart) were incubated at high temperature, followed by construction of 16S rRNA gene amplicon libraries (V3-V4 region; Illumina MiSeq) revealing enrichment of species-level thermospore OTUs. A sulfate reducing bacterium from site EGM080 was purified and classified based on its rRNA gene sequence as Desulfotomaculum geothermicum. Prior to thermospore staining the culture was kept in the death/ decline phase for 16 weeks to promote sporulation. Samples of D. geothermicum and the source marine sediment were fixed, stained then analysed using brightfield, phase contrast or fluorescence microscopy. Thermospores in pure culture were identified using phase contrast but were difficult to observe in the sediment sample due to particle aggregation. The Schaeffer-Fulton technique aided thermospore identification in a complex sediment sample matrix as thermospores were stained bright green, and also revealed that there were only spores and no (red stained) vegetative cells in the culture. Treatment with DAPI gave dull fluorescing cells but also provided insight into the behaviour of thermospores in sediment suspensions. Spores in the culture medium were free floating but

  6. An integrated lab-on-chip for rapid identification and simultaneous differentiation of tropical pathogens.

    Directory of Open Access Journals (Sweden)

    Jeslin J L Tan

    Full Text Available Tropical pathogens often cause febrile illnesses in humans and are responsible for considerable morbidity and mortality. The similarities in clinical symptoms provoked by these pathogens make diagnosis difficult. Thus, early, rapid and accurate diagnosis will be crucial in patient management and in the control of these diseases. In this study, a microfluidic lab-on-chip integrating multiplex molecular amplification and DNA microarray hybridization was developed for simultaneous detection and species differentiation of 26 globally important tropical pathogens. The analytical performance of the lab-on-chip for each pathogen ranged from 102 to 103 DNA or RNA copies. Assay performance was further verified with human whole blood spiked with Plasmodium falciparum and Chikungunya virus that yielded a range of detection from 200 to 4×105 parasites, and from 250 to 4×107 PFU respectively. This lab-on-chip was subsequently assessed and evaluated using 170 retrospective patient specimens in Singapore and Thailand. The lab-on-chip had a detection sensitivity of 83.1% and a specificity of 100% for P. falciparum; a sensitivity of 91.3% and a specificity of 99.3% for P. vivax; a positive 90.0% agreement and a specificity of 100% for Chikungunya virus; and a positive 85.0% agreement and a specificity of 100% for Dengue virus serotype 3 with reference methods conducted on the samples. Results suggested the practicality of an amplification microarray-based approach in a field setting for high-throughput detection and identification of tropical pathogens.

  7. Identification of a novel COL1A1 frameshift mutation, c.700delG, in a Chinese osteogenesis imperfecta family

    Science.gov (United States)

    Wang, Xiran; Pei, Yu; Dou, Jingtao; Lu, Juming; Li, Jian; Lv, Zhaohui

    2015-01-01

    Osteogenesis imperfecta (OI) is a family of genetic disorders associated with bone loss and fragility. Mutations associated with OI have been found in genes encoding the type I collagen chains. People with OI type I often produce insufficient α1-chain type I collagen because of frameshift, nonsense, or splice site mutations in COL1A1 or COL1A2. This report is of a Chinese daughter and mother who had both experienced two bone fractures. Because skeletal fragility is predominantly inherited, we focused on identifying mutations in COL1A1 and COL1A2 genes. A novel mutation in COL1A1, c.700delG, was detected by genomic DNA sequencing in the mother and daughter, but not in their relatives. The identification of this mutation led to the conclusion that they were affected by mild OI type I. Open reading frame analysis indicated that this frameshift mutation would truncate α1-chain type I collagen at residue p263 (p.E234KfsX264), while the wild-type protein would contain 1,464 residues. The clinical data were consistent with the patients’ diagnosis of mild OI type I caused by haploinsufficiency of α1-chain type I collagen. Combined with previous reports, identification of the novel mutation COL1A1-c.700delG in these patients suggests that additional genetic and environmental factors may influence the severity of OI. PMID:25983617

  8. [Rapid detection of hot spot mutations of FGFR3 gene with PCR-high resolution melting assay].

    Science.gov (United States)

    Li, Shan; Wang, Han; Su, Hua; Gao, Jinsong; Zhao, Xiuli

    2017-08-10

    To identify the causative mutations in five individuals affected with dyschondroplasia and develop an efficient procedure for detecting hot spot mutations of the FGFR3 gene. Genomic DNA was extracted from peripheral blood samples with a standard phenol/chloroform method. PCR-Sanger sequencing was used to analyze the causative mutations in the five probands. PCR-high resolution melting (HRM) was developed to detect the identified mutations. A c.1138G>A mutation in exon 8 was found in 4 probands, while a c.1620C>G mutation was found in exon 11 of proband 5 whom had a mild phenotype. All patients were successfully distinguished from healthy controls with the PCR-HRM method. The results of HRM analysis were highly consistent with that of Sanger sequencing. The Gly380Arg and Asn540Lys are hot spot mutations of the FGFR3 gene among patients with ACH/HCH. PCR-HRM analysis is more efficient for detecting hot spot mutations of the FGFR3 gene.

  9. Identification of syncytial mutations in a clinical isolate of herpes simplex virus 2

    International Nuclear Information System (INIS)

    Muggeridge, Martin I.; Grantham, Michael L.; Johnson, F. Brent

    2004-01-01

    Small polykaryocytes resulting from cell fusion are found in herpes simplex virus (HSV) lesions in patients, but their significance for viral spread and pathogenesis is unclear. Although syncytial variants causing extensive fusion in tissue culture can be readily isolated from laboratory strains, they are rarely found in clinical isolates, suggesting that extensive cell fusion may be deleterious in vivo. Syncytial mutations have previously been identified for several laboratory strains, but not for clinical isolates of HSV type 2. To address this deficiency, we studied a recent syncytial clinical isolate, finding it to be a mixture of two syncytial and one nonsyncytial strain. The two syncytial strains have novel mutations in glycoprotein B, and in vitro cell fusion assays confirmed that they are responsible for syncytium formation. This panel of clinical strains may be ideal for examining the effect of increased cell fusion on pathogenesis

  10. Molecular characterization of the llama FGF5 gene and identification of putative loss of function mutations.

    Science.gov (United States)

    Daverio, M S; Vidal-Rioja, L; Frank, E N; Di Rocco, F

    2017-12-01

    Llama, the most numerous domestic camelid in Argentina, has good fiber-production ability. Although a few genes related to other productive traits have been characterized, the molecular genetic basis of fiber growth control in camelids is still poorly understood. Fibroblast growth factor 5 (FGF5) is a secreted signaling protein that controls hair growth in humans and other mammals. Mutations in the FGF5 gene have been associated with long-hair phenotypes in several species. Here, we sequenced the llama FGF5 gene, which consists of three exons encoding 813 bp. cDNA analysis from hair follicles revealed the expression of two FGF5 alternative spliced transcripts, in one of which exon 2 is absent. DNA variation analysis showed four polymorphisms in the coding region: a synonymous SNP (c.210A>G), a single base deletion (c.348delA), a 12-bp insertion (c.351_352insCATATAACATAG) and a non-sense mutation (c.499C>T). The deletion was always found together with the insertion forming a haplotype and producing a putative truncated protein of 123 amino acids. The c.499C>T mutation also leads to a premature stop codon at position 168. In both cases, critical functional domains of FGF5, including one heparin binding site, are lost. All animals analyzed were homozygous for one of the deleterious mutations or compound heterozygous for both (i.e. c.348delA, c.351_352insCATATAACATAG/c.499T). Sequencing of guanaco samples showed that the FGF5 gene encodes a full-length 270-amino acid protein. These results suggest that FGF5 is likely functional in short-haired wild species and non-functional in the domestic fiber-producing species, the llama. © 2017 Stichting International Foundation for Animal Genetics.

  11. Congenital sucrase-isomaltase deficiency: identification of a common Inuit founder mutation.

    Science.gov (United States)

    Marcadier, Julien L; Boland, Margaret; Scott, C Ronald; Issa, Kheirie; Wu, Zaining; McIntyre, Adam D; Hegele, Robert A; Geraghty, Michael T; Lines, Matthew A

    2015-02-03

    Congenital sucrase-isomaltase deficiency is a rare hereditary cause of chronic diarrhea in children. People with this condition lack the intestinal brush-border enzyme required for digestion of di- and oligosaccharides, including sucrose and isomaltose, leading to malabsorption. Although the condition is known to be highly prevalent (about 5%-10%) in several Inuit populations, the genetic basis for this has not been described. We sought to identify a common mutation for congenital sucrase-isomaltase deficiency in the Inuit population. We sequenced the sucrase-isomaltase gene, SI, in a single Inuit proband with congenital sucrase-isomaltase deficiency who had severe fermentative diarrhea and failure to thrive. We then genotyped a further 128 anonymized Inuit controls from a variety of locales in the Canadian Arctic to assess for a possible founder effect. In the proband, we identified a novel, homozygous frameshift mutation, c.273_274delAG (p.Gly92Leufs*8), predicted to result in complete absence of a functional protein product. This change was very common among the Inuit controls, with an observed allele frequency of 17.2% (95% confidence interval [CI] 12.6%-21.8%). The predicted Hardy-Weinberg prevalence of congenital sucrase-isomaltase deficiency in Inuit people, based on this single founder allele, is 3.0% (95% CI 1.4%-4.5%), which is comparable with previous estimates. We found a common mutation, SI c.273_274delAG, to be responsible for the high prevalence of congenital sucrase-isomaltase deficiency among Inuit people. Targeted mutation testing for this allele should afford a simple and minimally invasive means of diagnosing this condition in Inuit patients with chronic diarrhea. © 2015 Canadian Medical Association or its licensors.

  12. Peptide nucleic acid probe-based fluorescence melting curve analysis for rapid screening of common JAK2, MPL, and CALR mutations.

    Science.gov (United States)

    Park, Joonhong; Song, Minsik; Jang, Woori; Chae, Hyojin; Lee, Gun Dong; Kim, KyungTak; Park, Heekyung; Kim, Myungshin; Kim, Yonggoo

    2017-02-01

    We developed and evaluated the feasibility of peptide nucleic acid (PNA)-based fluorescence melting curve analysis (FMCA) to detect common mutations in myeloproliferative neoplasms (MPNs). We have set up two separate reactions of PNA-based FMCA: JAK2 V617F &CALR p.Leu367fs*46 (set A) and MPL W515L/K &CALR p.Lys385fs*47 (set B). Clinical usefulness was validated with allele-specific real-time PCR, fragment analysis, Sanger sequencing in 57 BCR-ABL1-negative MPNs. The limit of detection (LOD) of PNA-based FMCA was approximately 10% for each mutation and interference reactions using mixtures of different mutations were not observed. Non-specific amplification was not observed in normal control. PNA-based FMCA was able to detect all JAK2 V617F (n=20), CALR p.Leu367fs*46 (n=10) and p.Lys385fs*47 (n=8). Three of six MPL mutations were detected except three samples with low mutant concentration in out of LOD. JAK2 exon 12 mutations (n=7) were negative without influencing V617F results. Among six variant CALR exon 9 mutations, two were detected by this method owing to invading of probe binding site. PNA-based FMCA for detecting common JAK2, MPL, and CALR mutations is a rapid, simple, and sensitive technique in BCR-ABL1-negative MPNs with >10% mutant allele at the time of initial diagnosis. Copyright © 2016 Elsevier B.V. All rights reserved.

  13. PCR-Restriction Fragment Length Polymorphism for Rapid, Low-Cost Identification of Isoniazid-Resistant Mycobacterium tuberculosis▿

    Science.gov (United States)

    Caws, Maxine; Tho, Dau Quang; Duy, Phan Minh; Lan, Nguyen Thi Ngoc; Hoa, Dai Viet; Torok, Mili Estee; Chau, Tran Thi Hong; Van Vinh Chau, Nguyen; Chinh, Nguyen Tran; Farrar, Jeremy

    2007-01-01

    PCR-restriction fragment length poymorphism (PCR-RFLP) is a simple, robust technique for the rapid identification of isoniazid-resistant Mycobacterium tuberculosis. One hundred consecutive isolates from a Vietnamese tuberculosis hospital were tested by MspA1I PCR-RFLP for the detection of isoniazid-resistant katG_315 mutants. The test had a sensitivity of 80% and a specificity of 100% against conventional phenotypic drug susceptibility testing. The positive and negative predictive values were 1 and 0.86, respectively. None of the discrepant isolates had mutant katG_315 codons by sequencing. The test is cheap (less than $1.50 per test), specific, and suitable for the rapid identification of isoniazid resistance in regions with a high prevalence of katG_315 mutants among isoniazid-resistant M. tuberculosis isolates. PMID:17428939

  14. Identification of a novel NHS mutation in a Chinese family with Nance-Horan syndrome.

    Science.gov (United States)

    Li, Aijun; Li, Bingzhen; Wu, Lemeng; Yang, Liping; Chen, Ningning; Ma, Zhizhong

    2015-04-01

    To identiy the disease causing mutation in a Chinese family presenting with early-onset cataract and dental anomalies. A specific Hereditary Eye Disease Enrichment Panel (HEDEP) (personalized customization by MyGenostics, Baltimore, MD) based on targeted exome capture technology was used to collect the protein coding regions of 30 early-onset cataract associated genes, and high throughput sequencing was done with Illumina HiSeq 2000 platform. The identified variant was confirmed with Sanger sequencing. A novel deletion in exon 4 (c.852delG) of NHS gene was identified; the identified 1 bp deletion altered the reading frame and was predicted to result in a premature stop codon after the addition of twelve novel amino acid (p.S285PfsX13). This mutation co-segregated in affected males and obligate female carriers, but was absent in 100 matched controls. Our findings broaden the spectrum of NHS mutations causing Nance-Horan syndrome and phenotypic spectrum of the disease in Chinese patients.

  15. Identification of a Novel GLA Mutation (L206 P) in a Patient with Fabry Disease.

    Science.gov (United States)

    Kim, Ji-Hoon; Kim, Gee-Hee; Park, Hoon-Suk; Choi, Jin-A; Bae, Jung-Min; Cho, Uiju

    2017-03-01

    We report a new α-Galactosidase A (αGal-A) mutation in a 39-year-old Korean born, male Fabry disease patient. Fabry disease is a devastating, progressive inborn error of metabolism caused by X-linked genetic mutations. In this case, the first clinical symptom to occur was in childhood consisting of a burning pain originating in the extremities then radiating inwards to the limbs. This patient also stated to have ringing in his ears, angiokeratomas on his trunk, and cornea verticillata. He visited an outpatient cardiologist due to intermittent and atypical chest discomfort at the age of 39. Electrocardiographic and echocardiographic examination showed left ventricular hypertrophy. A physical examination revealed proteinuria without hematuria. The patient's plasma αGal-A activity was markedly lower than the mean value of the controls. After genetic counseling and obtaining written informed consent, we identified one hemizygous mutation in exon 4 of galactosidase alpha, c.617T>C (p.Leu206 Pro). He was eventually diagnosed as having Fabry disease.

  16. Identification of fibrillin 1 gene mutations in patients with bicuspid aortic valve (BAV) without Marfan syndrome

    Science.gov (United States)

    2014-01-01

    Background Bicuspid aortic valve (BAV) is the most frequent congenital heart disease with frequent involvement in thoracic aortic dilatation, aneurysm and dissection. Although BAV and Marfan syndrome (MFS) share some clinical features, and some MFS patients with BAV display mutations in FBN1, the gene encoding fibrillin-1, the genetic background of isolated BAV is poorly defined. Methods Ten consecutive BAV patients [8 men, age range 24–42 years] without MFS were clinically characterized. BAV phenotype and function, together with evaluation of aortic morphology, were comprehensively assessed by Doppler echocardiography. Direct sequencing of each FBN1 exon with flanking intron sequences was performed on eight patients. Results We detected three FBN1 mutations in two patients (aged 24 and 25 years) displaying aortic root aneurysm ≥50 mm and moderate aortic regurgitation. In particular, one patient had two mutations (p.Arg2726Trp and p.Arg636Gly) one of which has been previously associated with variable Marfanoid phenotypes. The other patient showed a pArg529Gln substitution reported to be associated with an incomplete MFS phenotype. Conclusions The present findings enlarge the clinical spectrum of isolated BAV to include patients with BAV without MFS who have involvement of FBN1 gene. These results underscore the importance of accurate phenotyping of BAV aortopathy and of clinical characterization of BAV patients, including investigation of systemic connective tissue manifestations and genetic testing. PMID:24564502

  17. Fanconi anemia in Tunisia: high prevalence of group A and identification of new FANCA mutations.

    Science.gov (United States)

    Bouchlaka, Chiraz; Abdelhak, Sonia; Amouri, Ahlem; Ben Abid, Hela; Hadiji, Sondes; Frikha, Mounir; Ben Othman, Tarek; Amri, Fethi; Ayadi, Hammadi; Hachicha, Mongia; Rebaï, Ahmed; Saad, Ali; Dellagi, Koussay

    2003-01-01

    Fanconi anemia (FA) is a rare autosomal recessive disease characterized by progressive pancytopenia, congenital malformations, and predisposition to acute myeloid leukemia. Fanconi anemia is genetically heterogeneous, with at least eight distinct complementation groups of FA (A, B, C, D1, D2, E, F, and G) having been defined by somatic cell fusion studies. Six genes (FANCA, FANCC, FANCD2, FANCE, FANCG, and FANCF) have been cloned. Mutations of the seventh Fanconi anemia gene, BRCA2, have been shown to lead to FAD1 and probably FAB groups. In order to characterize the molecular defects underlying FA in Tunisia, 39 families were genotyped with microsatellite markers linked to known FA gene. Haplotype analysis and homozygosity mapping assigned 43 patients belonging to 34 families to the FAA group, whereas one family was probably not linked to the FANCA gene or to any known FA genes. For patients belonging to the FAA group, screening for mutations revealed four novel mutations: two small homozygous deletions 1693delT and 1751-1754del, which occurred in exon 17 and exon 19, respectively, and two transitions, viz., 513G-->A in exon 5 and A-->G at position 166 (IVS24+166A-->G) of intron 24. Two new polymorphisms were also identified in intron 24 (IVS24-5G/A and IVS24-6C/G).

  18. The use of newly developed real-time PCR for the rapid identification of bacteria in culture-negative osteomyelitis.

    Science.gov (United States)

    Kobayashi, Naomi; Bauer, Thomas W; Sakai, Hiroshige; Togawa, Daisuke; Lieberman, Isador H; Fujishiro, Takaaki; Procop, Gary W

    2006-12-01

    We report a case of a culture-negative osteomyelitis in which our newly developed real-time polymerase chain reaction (PCR) could differentiate Staphylococcus aureus from Staphylococcus epidermidis. This is the first report that described the application of this novel assay to an orthopedics clinical sample. This assay may be useful for other clinical culture-negative cases in a combination with a broad-spectrum assay as a rapid microorganism identification method.

  19. Rapid identification of ascomycetous yeasts from clinical specimens by a molecular method based on flow cytometry and comparison with identifications from phenotypic assays.

    Science.gov (United States)

    Page, Brent T; Shields, Christine E; Merz, William G; Kurtzman, Cletus P

    2006-09-01

    This study was designed to compare the identification of ascomycetous yeasts recovered from clinical specimens by using phenotypic assays (PA) and a molecular flow cytometric (FC) method. Large-subunit rRNA domains 1 and 2 (D1/D2) gene sequence analysis was also performed and served as the reference for correct strain identification. A panel of 88 clinical isolates was tested that included representatives of nine commonly encountered species and six infrequently encountered species. The PA included germ tube production, fermentation of seven carbohydrates, morphology on corn meal agar, urease and phenoloxidase activities, and carbohydrate assimilation tests when needed. The FC method (Luminex) employed species-specific oligonucleotides attached to polystyrene beads, which were hybridized with D1/D2 amplicons from the unidentified isolates. The PA identified 81 of 88 strains correctly but misidentified 4 of Candida dubliniensis, 1 of C. bovina, 1 of C. palmioleophila, and 1 of C. bracarensis. The FC method correctly identified 79 of 88 strains and did not misidentify any isolate but did not identify nine isolates because oligonucleotide probes were not available in the current library. The FC assay takes approximately 5 h, whereas the PA takes from 2 h to 5 days for identification. In conclusion, PA did well with the commonly encountered species, was not accurate for uncommon species, and takes significantly longer than the FC method. These data strongly support the potential of FC technology for rapid and accurate identification of medically important yeasts. With the introduction of new antifungals, rapid, accurate identification of pathogenic yeasts is more important than ever for guiding antifungal chemotherapy.

  20. Rapid identification of lettuce seed germination mutants by bulked segregant analysis and whole genome sequencing.

    Science.gov (United States)

    Huo, Heqiang; Henry, Isabelle M; Coppoolse, Eric R; Verhoef-Post, Miriam; Schut, Johan W; de Rooij, Han; Vogelaar, Aat; Joosen, Ronny V L; Woudenberg, Leo; Comai, Luca; Bradford, Kent J

    2016-11-01

    Lettuce (Lactuca sativa) seeds exhibit thermoinhibition, or failure to complete germination when imbibed at warm temperatures. Chemical mutagenesis was employed to develop lettuce lines that exhibit germination thermotolerance. Two independent thermotolerant lettuce seed mutant lines, TG01 and TG10, were generated through ethyl methanesulfonate mutagenesis. Genetic and physiological analyses indicated that these two mutations were allelic and recessive. To identify the causal gene(s), we applied bulked segregant analysis by whole genome sequencing. For each mutant, bulked DNA samples of segregating thermotolerant (mutant) seeds were sequenced and analyzed for homozygous single-nucleotide polymorphisms. Two independent candidate mutations were identified at different physical positions in the zeaxanthin epoxidase gene (ABSCISIC ACID DEFICIENT 1/ZEAXANTHIN EPOXIDASE, or ABA1/ZEP) in TG01 and TG10. The mutation in TG01 caused an amino acid replacement, whereas the mutation in TG10 resulted in alternative mRNA splicing. Endogenous abscisic acid contents were reduced in both mutants, and expression of the ABA1 gene from wild-type lettuce under its own promoter fully complemented the TG01 mutant. Conventional genetic mapping confirmed that the causal mutations were located near the ZEP/ABA1 gene, but the bulked segregant whole genome sequencing approach more efficiently identified the specific gene responsible for the phenotype. © 2016 The Authors The Plant Journal © 2016 John Wiley & Sons Ltd.

  1. Identification of novel peptide ligands for the cancer-specific receptor mutation EFGRvIII using a mixture-based synthetic combinatorial library

    DEFF Research Database (Denmark)

    Denholt, Charlotte Lund; Hansen, Paul Robert; Pedersen, Nina

    2009-01-01

    We report here, the design and synthesis of a positional scanning synthetic combinatorial library for the identification of novel peptide ligands targeted against the cancer-specific epidermal growth factor tyrosine kinase receptor mutation variant III (EGFRvIII). This receptor is expressed in se...

  2. Identification of a mutation in ADD1/SREBP-1 in the spontaneously hypertensive rat

    Czech Academy of Sciences Publication Activity Database

    Pravenec, Michal; Jansa, Petr; Kostka, Vlastimil; Zídek, Václav; Křen, Vladimír; Forejt, Jiří; Kurtz, T. W.

    2001-01-01

    Roč. 12, č. 4 (2001), s. 295-298 ISSN 0938-8990 R&D Projects: GA ČR(CZ) GA305/00/1646; GA MŠk(CZ) LN00A079; GA ČR(CZ) GV204/98/K015 Grant - others:HHMI(US) 55000331 Institutional research plan: CEZ:AV0Z5011922 Keywords : mutations in genes * ADD1/SREBP-1c * spontaneously hypertensive rat Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 2.318, year: 2001

  3. The significance of gtf genes in caries expression: a rapid identification of Streptococcus mutans from dental plaque of child patients.

    Science.gov (United States)

    Mishra, Apurva; Pandey, Ramesh K; Manickam, Natesan

    2015-01-01

    Rapid phylogenetic and functional gene (gtfB) identification of S. mutans from the dental plaque derived from children. Dental plaque collected from fifteen patients of age group 7-12 underwent centrifugation followed by genomic DNA extraction for S. mutans. Genomic DNA was processed with S. mutans specific primers in suitable PCR condtions for phylogenetic and functional gene (gtfB) identification. The yield and results were confirmed by agarose gel electrophoresis. 1% agarose gel electrophoresis depicts the positive PCR amplification at 1,485 bp when compared with standard 1 kbp indicating the presence of S. mutans in the test sample. Another PCR reaction was set using gtfB primers specific for S. mutans for functional gene identification. 1.2% agarose gel electrophoresis was done and a positive amplication was observed at 192 bp when compared to 100 bp standards. With the advancement in molecular biology techniques, PCR based identification and quantification of the bacterial load can be done within hours using species-specific primers and DNA probes. Thus, this technique may reduce the laboratory time spend in conventional culture methods, reduces the possibility of colony identification errors and is more sensitive to culture techniques.

  4. Identification of the mutation causing progressive retinal atrophy in Old Danish Pointing Dog.

    Science.gov (United States)

    Karlskov-Mortensen, P; Proschowsky, H F; Gao, F; Fredholm, M

    2018-04-06

    Progressive retinal atrophy (PRA) is a common cause of blindness in many dog breeds. It is most often inherited as a simple Mendelian trait, but great genetic heterogeneity has been demonstrated both within and between breeds. In many breeds the genetic cause of the disease is not known, and until now, the Old Danish Pointing Dog (ODP) has been one of those breeds. ODP is one of the oldest dog breeds in Europe. Seventy years ago the breed almost vanished, but today a population still exists, primarily in Denmark but with some dogs in Germany and Sweden. PRA has been diagnosed in ODP since the late 1990s. It resembles late onset PRA in other dog breeds, and it is inherited as an autosomal recessive trait. In the present study, we performed whole-genome sequencing and identified a single base insertion (c.3149_3150insC) in exon 1 of C17H2orf71. This is the same mutation previously found to cause PRA in Gordon Setters and Irish Setters, and it was later found in Tibetan Terrier, Standard Poodle and the Polski Owczarek Nizinny. The presence of the mutation in such a diverse range of breeds indicates an origin preceding creation of modern dog breeds. Hence, we screened 262 dogs from 44 different breeds plus four crossbred dogs, and can subsequently add Miniature Poodle and another polish sheepdog, the Polski Owczarek Podhalanski, to the list of affected breeds. © 2018 Stichting International Foundation for Animal Genetics.

  5. An immuno-wall microdevice exhibits rapid and sensitive detection of IDH1-R132H mutation specific to grade II and III gliomas

    Science.gov (United States)

    Yamamichi, Akane; Kasama, Toshihiro; Ohka, Fumiharu; Suzuki, Hiromichi; Kato, Akira; Motomura, Kazuya; Hirano, Masaki; Ranjit, Melissa; Chalise, Lushun; Kurimoto, Michihiro; Kondo, Goro; Aoki, Kosuke; Kaji, Noritada; Tokeshi, Manabu; Matsubara, Toshio; Senga, Takeshi; Kaneko, Mika K.; Suzuki, Hidenori; Hara, Masahito; Wakabayashi, Toshihiko; Baba, Yoshinobu; Kato, Yukinari; Natsume, Atsushi

    2016-01-01

    World Health Organization grade II and III gliomas most frequently occur in the central nervous system (CNS) in adults. Gliomas are not circumscribed; tumor edges are irregular and consist of tumor cells, normal brain tissue, and hyperplastic reactive glial cells. Therefore, the tumors are not fully resectable, resulting in recurrence, malignant progression, and eventual death. Approximately 69-80% of grade II and III gliomas harbor mutations in the isocitrate dehydrogenase 1 gene (IDH1), of which 83-90% are found to be the IDH1-R132H mutation. Detection of the IDH1-R132H mutation should help in the differential diagnosis of grade II and III gliomas from other types of CNS tumors and help determine the boundary between the tumor and normal brain tissue. In this study, we established a highly sensitive antibody-based device, referred to as the immuno-wall, to detect the IDH1-R132H mutation in gliomas. The immuno-wall causes an immunoreaction in microchannels fabricated using a photo-polymerizing polymer. This microdevice enables the analysis of the IDH1 status with a small sample within 15 min with substantially high sensitivity. Our results suggested that 10% content of the IDH1-R132H mutation in a sample of 0.33 μl volume, with 500 ng protein, or from 500 cells is theoretically sufficient for the analysis. The immuno-wall device will enable the rapid and highly sensitive detection of the IDH1-R132H mutation in routine clinical practice.

  6. Rapid point-of-care testing for epidermal growth factor receptor gene mutations in patients with lung cancer using cell-free DNA from cytology specimen supernatants.

    Science.gov (United States)

    Asaka, Shiho; Yoshizawa, Akihiko; Saito, Kazusa; Kobayashi, Yukihiro; Yamamoto, Hiroshi; Negishi, Tatsuya; Nakata, Rie; Matsuda, Kazuyuki; Yamaguchi, Akemi; Honda, Takayuki

    2018-06-01

    Epidermal growth factor receptor (EGFR) mutations are associated with responses to EGFR tyrosine kinase inhibitors (EGFR-TKIs) in non-small-cell lung cancer (NSCLC). Our previous study revealed a rapid point-of-care system for detecting EGFR mutations. This system analyzes cell pellets from cytology specimens using droplet-polymerase chain reaction (d-PCR), and has a reaction time of 10 min. The present study aimed to validate the performance of the EGFR d-PCR assay using cell-free DNA (cfDNA) from supernatants obtained from cytology specimens. Assay results from cfDNA supernatant analyses were compared with those from cell pellets for 90 patients who were clinically diagnosed with, or suspected of having, lung cancer (80 bronchial lavage fluid samples, nine pleural effusion samples and one spinal fluid sample). EGFR mutations were identified in 12 and 15 cases using cfDNA supernatants and cell pellets, respectively. The concordance rates between cfDNA-supernatant and cell‑pellet assay results were 96.7% [kappa coefficient (K)=0.87], 98.9% (K=0.94), 98.9% (K=0.79) and 98.9% (K=0.79) for total EGFR mutations, L858R, E746_A750del and T790M, respectively. All 15 patients with EGFR mutation-positive results, as determined by EGFR d-PCR assay using cfDNA supernatants or cell pellets, also displayed positive results by conventional EGFR assays using tumor tissue or cytology specimens. Notably, EGFR mutations were even detected in five cfDNA supernatants for which the cytological diagnoses of the corresponding cell pellets were 'suspicious for malignancy', 'atypical' or 'negative for malignancy.' In conclusion, this rapid point-of-care system may be considered a promising novel screening method that may enable patients with NSCLC to receive EGFR-TKI therapy more rapidly, whilst also reserving cell pellets for additional morphological and molecular analyses.

  7. Rapid Identification of Emerging Human-Pathogenic Sporothrix Species with Rolling Circle Amplification

    NARCIS (Netherlands)

    Rodrigues, Anderson M; Najafzadeh, Mohammad J; de Hoog, G Sybren; de Camargo, Zoilo P

    2015-01-01

    Sporothrix infections are emerging as an important human and animal threat among otherwise healthy patients, especially in Brazil and China. Correct identification of sporotrichosis agents is beneficial for epidemiological surveillance, enabling implementation of adequate public-health policies and

  8. Exploring MALDI-TOF MS approach for a rapid identification of Mycobacterium avium ssp. paratuberculosis field isolates.

    Science.gov (United States)

    Ricchi, M; Mazzarelli, A; Piscini, A; Di Caro, A; Cannas, A; Leo, S; Russo, S; Arrigoni, N

    2017-03-01

    The aim of the study was to explore the suitability of matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry (MALDI-TOF MS) for a rapid and correct identification of Mycobacterium avium ssp. paratuberculosis (MAP) field isolates. MALDI-TOF MS approach is becoming one of the most popular tests for the identification of intact bacterial cells which has been shown to be fast and reliable. For this purpose, 36 MAP field isolates were analysed through MALDI-TOF MS and the spectra compared with two different databases: one provided by the vendor of the system employed (Biotyper ver. 3·0; Bruker Daltonics) and a homemade database containing spectra from both tuberculous and nontuberculous Mycobacteria. Moreover, principal component analysis procedure was employed to confirm the ability of MALDI-TOF MS to discriminate between very closely related subspecies. Our results suggest MAP can be differentiated from other Mycobacterium species, both when the species are very close (M. intracellulare) and when belonging to different subspecies (M. avium ssp. avium and M. avium ssp. silvaticum). The procedure applied is fast, easy to perform, and achieves an earlier accurate species identification of MAP and nontuberculous Mycobacteria in comparison to other procedures. The gold standard test for the diagnosis of paratuberculosis is still isolation of MAP by cultural methods, but additional assays, such as qPCR and subculturing for determination of mycobactin dependency are required to confirm its identification. We have provided here evidence pertaining to the usefulness of MALDI-TOF MS approach for a rapid identification of this mycobacterium among other members of M. avium complex. © 2016 The Society for Applied Microbiology.

  9. Clinical laboratory evaluation of the Auto-Microbic system for rapid identification of Enterobacteriaceae.

    OpenAIRE

    Hasyn, J J; Cundy, K R; Dietz, C C; Wong, W

    1981-01-01

    The capability of the Auto-Microbic system (Vitek Systems, Inc., Hazelwood, Mo.) has been expanded to identify members of the family Enterobacteriaceae with the use of a sealed, disposable accessory card (the Enterobacteriaceae Biochemical Card) containing 26 biochemical tests. To judge the accuracy of the AutoMicrobic system's identification in a hospital laboratory, 933 Enterobacteriaceae isolates were studied. The AutoMicrobic system provided the correct identification for 905 of the isola...

  10. Development of a new protocol for rapid bacterial identification and susceptibility testing directly from urine samples.

    Science.gov (United States)

    Zboromyrska, Y; Rubio, E; Alejo, I; Vergara, A; Mons, A; Campo, I; Bosch, J; Marco, F; Vila, J

    2016-06-01

    The current gold standard method for the diagnosis of urinary tract infections (UTI) is urine culture that requires 18-48 h for the identification of the causative microorganisms and an additional 24 h until the results of antimicrobial susceptibility testing (AST) are available. The aim of this study was to shorten the time of urine sample processing by a combination of flow cytometry for screening and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS) for bacterial identification followed by AST directly from urine. The study was divided into two parts. During the first part, 675 urine samples were processed by a flow cytometry device and a cut-off value of bacterial count was determined to select samples for direct identification by MALDI-TOF-MS at ≥5 × 10(6) bacteria/mL. During the second part, 163 of 1029 processed samples reached the cut-off value. The sample preparation protocol for direct identification included two centrifugation and two washing steps. Direct AST was performed by the disc diffusion method if a reliable direct identification was obtained. Direct MALDI-TOF-MS identification was performed in 140 urine samples; 125 of the samples were positive by urine culture, 12 were contaminated and 3 were negative. Reliable direct identification was obtained in 108 (86.4%) of the 125 positive samples. AST was performed in 102 identified samples, and the results were fully concordant with the routine method among 83 monomicrobial infections. In conclusion, the turnaround time of the protocol described to diagnose UTI was about 1 h for microbial identification and 18-24 h for AST. Copyright © 2016 European Society of Clinical Microbiology and Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

  11. Identification of a homozygous PSTPIP1 mutation in a patient with a PAPA-like syndrome responding to canakinumab treatment.

    Science.gov (United States)

    Geusau, Alexandra; Mothes-Luksch, Nadine; Nahavandi, Hesam; Pickl, Winfried F; Wise, Carol A; Pourpak, Zahra; Ponweiser, Elisabeth; Eckhart, Leopold; Sunder-Plassmann, Raute

    2013-02-01

    Pyogenic sterile arthritis, pyoderma gangrenosum, and acne (PAPA) syndrome (OMIM 604416) is a rare autosomal dominant inherited autoinflammatory syndrome characterized by pyogenic sterile arthritis and less frequently accompanied by pyoderma gangrenosum and acne. It is associated with dominant missense mutations in the proline-serine-threonine phosphatase-interacting protein 1 gene (PSTPIP1) located on chromosome 15. The patient was diagnosed as having features of a PAPA-like syndrome in which cutaneous manifestations, such as pyoderma gangrenosum and acne fulminans, predominated. Sequencing of the PSTPIP1 gene was performed in the patient and his extended family. The patient's DNA analysis revealed a homozygous nucleotide exchange c.773G>C in the PSTPIP1 gene, leading to the substitution of glycine 258 by alanine (p.Gly258Ala), a previously reported heterozygous polymorphism. Heterozygous changes were identified in both of the patient's parents and in 7 other family members, all of whom were asymptomatic. The patient was treated with canakinumab, a human anti-interleukin 1β monoclonal antibody, which led to rapid remission of the symptoms. To our knowledge, this is the first reported case of the resolution of dermatological symptoms associated with a PAPA-like syndrome using canakinumab treatment. Further study of the p.Gly258Ala variant is warranted to determine whether this mutation has a role in causing an apparently recessive cutaneous syndrome resembling PAPA syndrome.

  12. NMR experiments for the rapid identification of P=O···H-X type hydrogen bonds in nucleic acids.

    Science.gov (United States)

    Duchardt-Ferner, Elke; Wöhnert, Jens

    2017-10-01

    Hydrogen bonds involving the backbone phosphate groups occur with high frequency in functional RNA molecules. They are often found in well-characterized tertiary structural motifs presenting powerful probes for the rapid identification of these motifs for structure elucidation purposes. We have shown recently that stable hydrogen bonds to the phosphate backbone can in principle be detected by relatively simple NMR-experiments, providing the identity of both the donor hydrogen and the acceptor phosphorous within the same experiment (Duchardt-Ferner et al., Angew Chem Int Ed Engl 50:7927-7930, 2011). However, for imino and hydroxyl hydrogen bond donor groups rapidly exchanging with the solvent as well as amino groups broadened by conformational exchange experimental sensitivity is severely hampered by extensive line broadening. Here, we present improved methods for the rapid identification of hydrogen bonds to phosphate groups in nucleic acids by NMR. The introduction of the SOFAST technique into 1 H, 31 P-correlation experiments as well as a BEST-HNP experiment exploiting 3h J N,P rather than 2h J H,P coupling constants enables the rapid and sensitive identification of these hydrogen bonds in RNA. The experiments are applicable for larger RNAs (up to ~ 100-nt), for donor groups influenced by conformational exchange processes such as amino groups and for hydrogen bonds with rather labile hydrogens such as 2'-OH groups as well as for moderate sample concentrations. Interestingly, the size of the through-hydrogen bond scalar coupling constants depends not only on the type of the donor group but also on the structural context. The largest coupling constants were measured for hydrogen bonds involving the imino groups of protonated cytosine nucleotides as donors.

  13. Enzymic colorimetry-based DNA chip: a rapid and accurate assay for detecting mutations for clarithromycin resistance in the 23S rRNA gene of Helicobacter pylori.

    Science.gov (United States)

    Xuan, Shi-Hai; Zhou, Yu-Gui; Shao, Bo; Cui, Ya-Lin; Li, Jian; Yin, Hong-Bo; Song, Xiao-Ping; Cong, Hui; Jing, Feng-Xiang; Jin, Qing-Hui; Wang, Hui-Min; Zhou, Jie

    2009-11-01

    Macrolide drugs, such as clarithromycin (CAM), are a key component of many combination therapies used to eradicate Helicobacter pylori. However, resistance to CAM is increasing in H. pylori and is becoming a serious problem in H. pylori eradication therapy. CAM resistance in H. pylori is mostly due to point mutations (A2142G/C, A2143G) in the peptidyltransferase-encoding region of the 23S rRNA gene. In this study an enzymic colorimetry-based DNA chip was developed to analyse single-nucleotide polymorphisms of the 23S rRNA gene to determine the prevalence of mutations in CAM-related resistance in H. pylori-positive patients. The results of the colorimetric DNA chip were confirmed by direct DNA sequencing. In 63 samples, the incidence of the A2143G mutation was 17.46 % (11/63). The results of the colorimetric DNA chip were concordant with DNA sequencing in 96.83 % of results (61/63). The colorimetric DNA chip could detect wild-type and mutant signals at every site, even at a DNA concentration of 1.53 x 10(2) copies microl(-1). Thus, the colorimetric DNA chip is a reliable assay for rapid and accurate detection of mutations in the 23S rRNA gene of H. pylori that lead to CAM-related resistance, directly from gastric tissues.

  14. Rapid mutation of Spirulina platensis by a new mutagenesis system of atmospheric and room temperature plasmas (ARTP and generation of a mutant library with diverse phenotypes.

    Directory of Open Access Journals (Sweden)

    Mingyue Fang

    Full Text Available In this paper, we aimed to improve the carbohydrate productivity of Spirulina platensis by generating mutants with increased carbohydrate content and growth rate. ARTP was used as a new mutagenesis tool to generate a mutant library of S. platensis with diverse phenotypes. Protocol for rapid mutation of S. platensis by 60 s treatment with helium driven ARTP and high throughput screening method of the mutants using the 96-well microplate and microplate reader was established. A mutant library of 62 mutants was then constructed and ideal mutants were selected out. The characteristics of the mutants after the mutagenesis inclined to be stable after around 9(th subculture, where the total mutation frequency and positive mutation frequency in terms of specific growth rate reached 45% and 25%, respectively. The mutants in mutant library showed diverse phenotypes in terms of cell growth rate, carbohydrate content and flocculation intensity. The positive mutation frequency in terms of cellular carbohydrate content with the increase by more than 20% percent than the wild strain was 32.3%. Compared with the wild strain, the representative mutants 3-A10 and 3-B2 showed 40.3% and 78.0% increase in carbohydrate content, respectively, while the mutant 4-B3 showed 10.5% increase in specific growth rate. The carbohydrate contents of the representative mutants were stable during different subcultures, indicating high genetic stability. ARTP was demonstrated to be an effective and non-GMO mutagenesis tool to generate the mutant library for multicellular microalgae.

  15. Limited copy number-high resolution melting (LCN-HRM) enables the detection and identification by sequencing of low level mutations in cancer biopsies.

    Science.gov (United States)

    Do, Hongdo; Dobrovic, Alexander

    2009-10-08

    Mutation detection in clinical tumour samples is challenging when the proportion of tumour cells, and thus mutant alleles, is low. The limited sensitivity of conventional sequencing necessitates the adoption of more sensitive approaches. High resolution melting (HRM) is more sensitive than sequencing but identification of the mutation is desirable, particularly when it is important to discriminate false positives due to PCR errors or template degradation from true mutations.We thus developed limited copy number - high resolution melting (LCN-HRM) which applies limiting dilution to HRM. Multiple replicate reactions with a limited number of target sequences per reaction allow low level mutations to be detected. The dilutions used (based on Ct values) are chosen such that mutations, if present, can be detected by the direct sequencing of amplicons with aberrant melting patterns. Using cell lines heterozygous for mutations, we found that the mutations were not readily detected when they comprised 10% of total alleles (20% tumour cells) by sequencing, whereas they were readily detectable at 5% total alleles by standard HRM. LCN-HRM allowed these mutations to be identified by direct sequencing of those positive reactions.LCN-HRM was then used to review formalin-fixed paraffin-embedded (FFPE) clinical samples showing discordant findings between sequencing and HRM for KRAS exon 2 and EGFR exons 19 and 21. Both true mutations present at low levels and sequence changes due to artefacts were detected by LCN-HRM. The use of high fidelity polymerases showed that the majority of the artefacts were derived from the damaged template rather than replication errors during amplification. LCN-HRM bridges the sensitivity gap between HRM and sequencing and is effective in distinguishing between artefacts and true mutations.

  16. Identification of uvrA gene mutation sites in two mitomycin-sensitive deinococcus radiodurans strains

    International Nuclear Information System (INIS)

    Du Zeji; Kong Xianrong

    1999-01-01

    Deinococcus radiodurans (Dr) possesses a prominent ability to repair the DNA injury induced by various DNA- damaging agents including mitomycin C(MC), ultraviolet light (UV) and ionizing radiation. DNA damage resistance was restored in MC sensitive (MC s ) mutants 2621 and 3021 by transforming with DNAs of four cosmids clones derived from the gene library of strain KD8301 which showed the property of wild type phenotype to DNA-damaging agents. Gene affected by mutation (mtcA or mtcB) in both mutants was cloned and its nucleotide sequence was determined. The deduced amino acid (aa) sequence of Dr uvrA gene product consists of 1016 aa and shares homology with many bacterial UvrA proteins. The mutation sites in both mutants were identified by analyzing the polymerase chain reaction (PCR) fragments derived from the genomic DNA of the mutants. A 144-base pairs (bp) deletion including the start codon for the uvr A gene was observed in DNA of the mutant 3021, causing a defect in the gene. On the other hand, an insertion sequence (IS) element intervened in the uvrA gene of the mutant 2621, suggesting the insertional inactivation of the gene. The IS element comprise 1322-bp long, flanked by 19-bp inverted terminal repeats (ITR), and generated a 6-bp target duplication (TD). Two open reading frames (ORF) were found in the IS element. The deduced aa sequences of large and small ORF show homology to a putative transposes found in IS4 of Escherichia coli (E. coli) and to a resolvent found in IS Xc5 of Xanthomonas campestris (Xc), respectively. This is the first discovery of IS element in deino-bacteria, and the IS element was designated IS2621

  17. Lncident: A Tool for Rapid Identification of Long Noncoding RNAs Utilizing Sequence Intrinsic Composition and Open Reading Frame Information

    Directory of Open Access Journals (Sweden)

    Siyu Han

    2016-01-01

    Full Text Available More and more studies have demonstrated that long noncoding RNAs (lncRNAs play critical roles in diversity of biological process and are also associated with various types of disease. How to rapidly identify lncRNAs and messenger RNA is the fundamental step to uncover the function of lncRNAs identification. Here, we present a novel method for rapid identification of lncRNAs utilizing sequence intrinsic composition features and open reading frame information based on support vector machine model, named as Lncident (LncRNAs identification. The 10-fold cross-validation and ROC curve are used to evaluate the performance of Lncident. The main advantage of Lncident is high speed without the loss of accuracy. Compared with the exiting popular tools, Lncident outperforms Coding-Potential Calculator, Coding-Potential Assessment Tool, Coding-Noncoding Index, and PLEK. Lncident is also much faster than Coding-Potential Calculator and Coding-Noncoding Index. Lncident presents an outstanding performance on microorganism, which offers a great application prospect to the analysis of microorganism. In addition, Lncident can be trained by users’ own collected data. Furthermore, R package and web server are simultaneously developed in order to maximize the convenience for the users. The R package “Lncident” can be easily installed on multiple operating system platforms, as long as R is supported.

  18. Rapid qualitative urinary tract infection pathogen identification by SeptiFast real-time PCR.

    Directory of Open Access Journals (Sweden)

    Lutz E Lehmann

    2011-02-01

    Full Text Available Urinary tract infections (UTI are frequent in outpatients. Fast pathogen identification is mandatory for shortening the time of discomfort and preventing serious complications. Urine culture needs up to 48 hours until pathogen identification. Consequently, the initial antibiotic regimen is empirical.To evaluate the feasibility of qualitative urine pathogen identification by a commercially available real-time PCR blood pathogen test (SeptiFast® and to compare the results with dipslide and microbiological culture.Pilot study with prospectively collected urine samples.University hospital.82 prospectively collected urine samples from 81 patients with suspected UTI were included. Dipslide urine culture was followed by microbiological pathogen identification in dipslide positive samples. In parallel, qualitative DNA based pathogen identification (SeptiFast® was performed in all samples.61 samples were SeptiFast® positive, whereas 67 samples were dipslide culture positive. The inter-methodological concordance of positive and negative findings in the gram+, gram- and fungi sector was 371/410 (90%, 477/492 (97% and 238/246 (97%, respectively. Sensitivity and specificity of the SeptiFast® test for the detection of an infection was 0.82 and 0.60, respectively. SeptiFast® pathogen identifications were available at least 43 hours prior to culture results.The SeptiFast® platform identified bacterial DNA in urine specimens considerably faster compared to conventional culture. For UTI diagnosis sensitivity and specificity is limited by its present qualitative setup which does not allow pathogen quantification. Future quantitative assays may hold promise for PCR based UTI pathogen identification as a supplementation of conventional culture methods.

  19. Rapid Classification and Identification of Multiple Microorganisms with Accurate Statistical Significance via High-Resolution Tandem Mass Spectrometry.

    Science.gov (United States)

    Alves, Gelio; Wang, Guanghui; Ogurtsov, Aleksey Y; Drake, Steven K; Gucek, Marjan; Sacks, David B; Yu, Yi-Kuo

    2018-06-05

    Rapid and accurate identification and classification of microorganisms is of paramount importance to public health and safety. With the advance of mass spectrometry (MS) technology, the speed of identification can be greatly improved. However, the increasing number of microbes sequenced is complicating correct microbial identification even in a simple sample due to the large number of candidates present. To properly untwine candidate microbes in samples containing one or more microbes, one needs to go beyond apparent morphology or simple "fingerprinting"; to correctly prioritize the candidate microbes, one needs to have accurate statistical significance in microbial identification. We meet these challenges by using peptide-centric representations of microbes to better separate them and by augmenting our earlier analysis method that yields accurate statistical significance. Here, we present an updated analysis workflow that uses tandem MS (MS/MS) spectra for microbial identification or classification. We have demonstrated, using 226 MS/MS publicly available data files (each containing from 2500 to nearly 100,000 MS/MS spectra) and 4000 additional MS/MS data files, that the updated workflow can correctly identify multiple microbes at the genus and often the species level for samples containing more than one microbe. We have also shown that the proposed workflow computes accurate statistical significances, i.e., E values for identified peptides and unified E values for identified microbes. Our updated analysis workflow MiCId, a freely available software for Microorganism Classification and Identification, is available for download at https://www.ncbi.nlm.nih.gov/CBBresearch/Yu/downloads.html . Graphical Abstract ᅟ.

  20. Rapid screening of guar gum using portable Raman spectral identification methods.

    Science.gov (United States)

    Srivastava, Hirsch K; Wolfgang, Steven; Rodriguez, Jason D

    2016-01-25

    Guar gum is a well-known inactive ingredient (excipient) used in a variety of oral pharmaceutical dosage forms as a thickener and stabilizer of suspensions and as a binder of powders. It is also widely used as a food ingredient in which case alternatives with similar properties, including chemically similar gums, are readily available. Recent supply shortages and price fluctuations have caused guar gum to come under increasing scrutiny for possible adulteration by substitution of cheaper alternatives. One way that the U.S. FDA is attempting to screen pharmaceutical ingredients at risk for adulteration or substitution is through field-deployable spectroscopic screening. Here we report a comprehensive approach to evaluate two field-deployable Raman methods--spectral correlation and principal component analysis--to differentiate guar gum from other gums. We report a comparison of the sensitivity of the spectroscopic screening methods with current compendial identification tests. The ability of the spectroscopic methods to perform unambiguous identification of guar gum compared to other gums makes them an enhanced surveillance alternative to the current compendial identification tests, which are largely subjective in nature. Our findings indicate that Raman spectral identification methods perform better than compendial identification methods and are able to distinguish guar gum from other gums with 100% accuracy for samples tested by spectral correlation and principal component analysis. Published by Elsevier B.V.

  1. Efficient detection of factor IX mutations by denaturing high-performance liquid chromatography in Taiwanese hemophilia B patients, and the identification of two novel mutations

    Directory of Open Access Journals (Sweden)

    Pei-Chin Lin

    2014-04-01

    Full Text Available Hemophilia B (HB is an X-linked recessive disorder characterized by mutations in the clotting factor IX (FIX gene that result in FIX deficiency. Previous studies have shown a wide variation of FIX gene mutations in HB. Although the quality of life in HB has greatly improved mainly because of prophylactic replacement therapy with FIX concentrates, there exists a significant burden on affected families and the medical care system. Accurate detection of FIX gene mutations is critical for genetic counseling and disease prevention in HB. In this study, we used denaturing high-performance liquid chromatography (DHPLC, which has proved to be a highly informative and practical means of detecting mutations, for the molecular diagnosis of our patients with HB. Ten Taiwanese families affected by HB were enrolled. We used the DHPLC technique followed by direct sequencing of suspected segments to detect FIX gene mutations. In all, 11 FIX gene mutations (8 point mutations, 2 small deletions/insertions, and 1 large deletion, including two novel mutations (exon6 c.687–695, del 9 mer and c.460–461, ins T were found. According to the HB pedigrees, 25% and 75% of our patients were defined as familial and sporadic HB cases, respectively. We show that DHPLC is a highly sensitive and cost-effective method for FIX gene analysis and can be used as a convenient system for disease prevention.

  2. Les huiles végétales au Vietnam : un secteur en mutation rapide

    Directory of Open Access Journals (Sweden)

    Benassi Jean-Louis

    2004-01-01

    Full Text Available Le secteur des huiles végétales au Vietnam, pays de 80 millions d’habitants au taux de croissance élevé, est en forte mutation. La consommation spécifique, encore faible (5 kg\\\\tête d’huiles et graisses, augmente rapidement. La production intérieure d’oléagineux étant limitée, les importations grimpent. Ceci vaut également pour les tourteaux. Le marché est largement dominé par des produits d’importation : huiles de palme et de soja d’un côté, tourteaux de soja de l’autre. L’huile est en majorité utilisée en alimentation directe, sous forme liquide. Une grande partie de l’offre embouteillée est constituée d’huiles de mélange, essentiellement à base de palme et de soja, mais du colza peut aussi être incorporé. Alors que la trituration est restée limitée, notamment du fait d’une production de copra déclinante, la capacité de raffinage s’est nettement développée ces dernières années, dans un contexte tarifaire favorable. Mais la situation est en train de changer : l’accord de libre échange de l’ASEAN prévoit une réduction des droits de douane entre pays membres devant conduire à des niveaux de protection très faibles d’ici à 2006. Face à l’augmentation de la demande, des questions importantes se posent aujourd’hui quant au développement de la production locale d’oléagineux pour limiter la dépendance aux importations et quant à la mise à niveau de l’industrie de transformation nationale, pour laquelle des projets existent déjà. Les principaux oléagineux nationaux sont actuellement le coco, l’arachide et le sésame. Surtout tournés vers l’exportation, ces produits ne peuvent contribuer que de façon limitée à l’approvisionnement du marché intérieur. Dans l’optique d’un développement de la production d’huile à partir de cultures locales, le soja a probablement un rôle majeur à jouer. Exploiter des ressources existantes est une autre possibilit

  3. Identification of a mutation associated with fatal Foal Immunodeficiency Syndrome in the Fell and Dales pony.

    Directory of Open Access Journals (Sweden)

    Laura Y Fox-Clipsham

    2011-07-01

    Full Text Available The Fell and Dales are rare native UK pony breeds at risk due to falling numbers, in-breeding, and inherited disease. Specifically, the lethal Mendelian recessive disease Foal Immunodeficiency Syndrome (FIS, which manifests as B-lymphocyte immunodeficiency and progressive anemia, is a substantial threat. A significant percentage (∼10% of the Fell ponies born each year dies from FIS, compromising the long-term survival of this breed. Moreover, the likely spread of FIS into other breeds is of major concern. Indeed, FIS was identified in the Dales pony, a related breed, during the course of this work. Using a stepwise approach comprising linkage and homozygosity mapping followed by haplotype analysis, we mapped the mutation using 14 FIS-affected, 17 obligate carriers, and 10 adults of unknown carrier status to a ∼1 Mb region (29.8 - 30.8 Mb on chromosome (ECA 26. A subsequent genome-wide association study identified two SNPs on ECA26 that showed genome-wide significance after Bonferroni correction for multiple testing: BIEC2-692674 at 29.804 Mb and BIEC2-693138 at 32.19 Mb. The associated region spanned 2.6 Mb from ∼29.6 Mb to 32.2 Mb on ECA26. Re-sequencing of this region identified a mutation in the sodium/myo-inositol cotransporter gene (SLC5A3; this causes a P446L substitution in the protein. This gene plays a crucial role in the regulatory response to osmotic stress that is essential in many tissues including lymphoid tissues and during early embryonic development. We propose that the amino acid substitution we identify here alters the function of SLC5A3, leading to erythropoiesis failure and compromise of the immune system. FIS is of significant biological interest as it is unique and is caused by a gene not previously associated with a mammalian disease. Having identified the associated gene, we are now able to eradicate FIS from equine populations by informed selective breeding.

  4. MALDI-TOF MS is more accurate than VITEK II ANC card and API Rapid ID 32 A system for the identification of Clostridium species.

    Science.gov (United States)

    Kim, Young Jin; Kim, Si Hyun; Park, Hyun-Jung; Park, Hae-Geun; Park, Dongchul; Song, Sae Am; Lee, Hee Joo; Yong, Dongeun; Choi, Jun Yong; Kook, Joong-Ki; Kim, Hye Ran; Shin, Jeong Hwan

    2016-08-01

    All 50 Clostridium difficile strains were definitely identified by Vitek2 system, Rapid ID 32A system, and MALDI-TOF. For 18 non-difficile Clostridium strains, the identification results were correct in 0, 2, and 17 strains by Vitek2, Rapid ID 32A, and MALDI-TOF, respectively. MALDI-TOF could be used as the primary tool for identification of Clostridium species. Copyright © 2016 Elsevier Ltd. All rights reserved.

  5. Rapid and reliable identification of Gram-negative bacteria and Gram-positive cocci by deposition of bacteria harvested from blood cultures onto the MALDI-TOF plate.

    OpenAIRE

    Barnini, S; Ghelardi, Emilia; Brucculeri, V; Morici, Paola; Lupetti, Antonella

    2015-01-01

    Background Rapid identification of the causative agent(s) of bloodstream infections using the matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) methodology can lead to increased empirical antimicrobial therapy appropriateness. Herein, we aimed at establishing an easier and simpler method, further referred to as the direct method, using bacteria harvested by serum separator tubes from positive blood cultures and placed onto the polished steel target plate for rapid identif...

  6. Rapid Sanger sequencing of the 16S rRNA gene for identification of some common pathogens.

    Directory of Open Access Journals (Sweden)

    Linxiang Chen

    Full Text Available Conventional Sanger sequencing remains time-consuming and laborious. In this study, we developed a rapid improved sequencing protocol of 16S rRNA for pathogens identification by using a new combination of SYBR Green I real-time PCR and Sanger sequencing with FTA® cards. To compare the sequencing quality of this method with conventional Sanger sequencing, 12 strains, including three kinds of strains (1 reference strain and 3 clinical strains, which were previously identified by biochemical tests, which have 4 Pseudomonas aeruginosa, 4 Staphyloccocus aureus and 4 Escherichia coli, were targeted. Additionally, to validate the sequencing results and bacteria identification, expanded specimens with 90 clinical strains, also comprised of the three kinds of strains which included 30 samples respectively, were performed as just described. The results showed that although statistical differences (P<0.05 were found in sequencing quality between the two methods, their identification results were all correct and consistent. The workload, the time consumption and the cost per batch were respectively light versus heavy, 8 h versus 11 h and $420 versus $400. In the 90 clinical strains, all of the Pseudomonas aeruginosa and Staphyloccocus aureus strains were correctly identified, but only 26.7% of the Escherichia coli strains were recognized as Escherichia coli, while 33.3% as Shigella sonnei and 40% as Shigella dysenteriae. The protocol described here is a rapid, reliable, stable and convenient method for 16S rRNA sequencing, and can be used for Pseudomonas aeruginosa and Staphyloccocus aureus identification, yet it is not completely suitable for discriminating Escherichia coli and Shigella strains.

  7. Comparison of Enzymatic Method Rapid Yeast Plus System with RFLP-PCR for Identification of Isolated Yeast from Vulvovaginal Candidiasis.

    Science.gov (United States)

    Hossein, Moallaei; Mirhendi, Seied Hossein; Brandão, João; Mirdashti, Reza; Rosado, Laura

    2011-09-01

    To compare two identification methods, i.e., restriction fragment length polymorphism (RFLP)-PCR analysis and enzymatic method Rapid TM Yeast Plus System to identify different species causing vulvovaginal candidiasis (VVC). Vaginal discharges of women who had attended the gynecology outpatient clinic of Mobini Hospital in Sabzevar, Iran were collected using cotton swabs and were cultured on Sabouraud dextrose agar. Isolated yeasts were identified by germ-tube testing and Rapid TM Yeast Plus System (Remel USA). For molecular identification, the isolated DNA was amplified with ITS1 and ITS4 universal primers and PCR products digested with the enzyme HpaІІ followed by agarose gel electrophoresis. Epidemiological and clinical features of women with respect to identified species were also evaluated. Out of 231 subjects enrolled, 62 VVC cases were detected. The isolated species were identified as follows: Candida albicans, 24 (38.7%), C. glabrata, 15 (24.2%), C. kefyr, 13 (21.0%) C. krusei, 9 (14.5%), and Saccharomyces cerevisiae, 1 (1.6%) by RFLP-PCR method; whereas findings by Rapid TM Yeast Plus System were C. albicans, 24 (38.7%), C. glabrata, 5 (8%), C. kefyr, 11 (17.7%) C. krusei, 2 (3.2%), S. cerevisiae, 9 (14.5%), and C. tropicalis, 6 (9.6%) as well as other nonpathogenic yeasts, 4 (6.9%). Statistical comparison showed that there is no significant difference in identification of C. albicans by the two methods; although, in this study, it was not true about other species of yeasts. A correlation between clinical and laboratory findings is important as it enables us to administer an appropriate treatment on time.

  8. Rapid identification of genes controlling virulence and immunity in malaria parasites

    KAUST Repository

    Abkallo, Hussein M.; Martinelli, Axel; Inoue, Megumi; Ramaprasad, Abhinay; Xangsayarath, Phonepadith; Gitaka, Jesse; Tang, Jianxia; Yahata, Kazuhide; Zoungrana, Augustin; Mitaka, Hayato; Acharjee, Arita; Datta, Partha P.; Hunt, Paul; Carter, Richard; Kaneko, Osamu; Mustonen, Ville; Illingworth, Christopher J. R.; Pain, Arnab; Culleton, Richard

    2017-01-01

    Identifying the genetic determinants of phenotypes that impact disease severity is of fundamental importance for the design of new interventions against malaria. Here we present a rapid genome-wide approach capable of identifying multiple genetic

  9. Electron microscopy of rapid identification of animal viruses in hematoxylin-eosin sections.

    Science.gov (United States)

    Bhatnagar, R; Johnson, G R; Christian, R G

    1977-01-01

    Routine hematoxylin-eosin stained, paraffin sections were processed for electron microscopy, using a rapid method for localization of animal viruses. Formalin fixation was effective in preserving DNA as well as RNA viruses, however cellular fine structural details and organelles were not well preserved. The procedure is useful for morphological recognition of viral groups and as a rapid diagnostic aid for identifying viral disease. Images Fig. 1. Fig. 2. Fig. 3. Fig. 4. Fig. 5. Fig. 6. PMID:72592

  10. Rapid and accurate identification of isolates of Candida species by melting peak and melting curve analysis of the internally transcribed spacer region 2 fragment (ITS2-MCA)

    NARCIS (Netherlands)

    Decat, E.; van Mechelen, E.; Saerens, B.; Vermeulen, S.J.T.; Boekhout, T.; de Blaiser, S.; Vaneechoutte, M.; Deschaght, P.

    2013-01-01

    Rapid identification of clinically important yeasts can facilitate the initiation of anti-fungal therapy, since susceptibility is largely species-dependent. We evaluated melting peak and melting curve analysis of the internally transcribed spacer region 2 fragment (ITS2-MCA) as an identification

  11. Rapid identification of clinical members of Fusarium fujikuroi complex using MALDI-TOF MS

    NARCIS (Netherlands)

    Al-Hatmi, Abdullah Ms; Normand, Anne-Cécile; van Diepeningen, Anne D; Hendrickx, Marijke; de Hoog, G Sybren; Piarroux, Renaud

    2015-01-01

    AIM: To develop the matrix-assisted laser desorption ionization mass spectrometry (MALDI-TOF MS) method for identification of Fusarium species within Fusarium fujikuroi complex for use in clinical microbiology laboratories. MATERIALS & METHODS: A total of 24 reference and 60 clinical and

  12. Rapid detection of drug resistance and mutational patterns of extensively drug-resistant strains by a novel GenoType® MTBDRsl assay

    Directory of Open Access Journals (Sweden)

    A K Singh

    2013-01-01

    Full Text Available Background: The emergence of extensively drug-resistant tuberculosis (XDR-TB is a major concern in the India. The burden of XDR-TB is increasing due to inadequate monitoring, lack of proper diagnosis, and treatment. The GenoType ® Mycobacterium tuberculosis drug resistance second line (MTBDRsl assay is a novel line probe assay used for the rapid detection of mutational patterns conferring resistance to XDR-TB. Aim: The aim of this study was to study the rapid detection of drug resistance and mutational patterns of the XDR-TB by a novel GenoType ® MTBDRsl assay. Materials and Methods: We evaluated 98 multidrug-resistant (MDR M. tuberculosis isolates for second line drugs susceptibility testing by 1% proportion method (BacT/ALERT 3D system and GenoType ® MTBDRsl assay for rapid detection of conferring drug resistance to XDR-TB. Results: A total of seven (17.4% were identified as XDR-TB by using standard phenotypic method. The concordance between phenotypic and GenoType ® MTBDRsl assay was 91.7-100% for different antibiotics. The sensitivity and specificity of the MTBDRsl assay were 100% and 100% for aminoglycosides; 100% and 100% for fluoroquinolones; 91.7% and 100% for ethambutol. The most frequent mutations and patterns were gyrA MUT1 (A90V in seven (41.2% and gyrA + WT1-3 + MUT1 in four (23.5%; rrs MUT1 (A1401G in 11 (64.7%, and rrs WT1-2 + MUT1 in eight (47.1%; and embB MUT1B (M306V in 11 (64.7% strains. Conclusions: These data suggest that the GenoType ® MTBDRsl assay is rapid, novel test for detection of resistance to second line anti-tubercular drugs. This assay provides additional information about the frequency and mutational patterns responsible for XDR-TB resistance.

  13. Identification of the Related Substances in Ampicillin Capsule by Rapid Resolution Liquid Chromatography Coupled with Electrospray Ionization Tandem Mass Spectrometry

    Directory of Open Access Journals (Sweden)

    Lei Zhang

    2014-01-01

    Full Text Available Rapid Resolution Liquid Chromatography coupled with Electrospray Ionization Tandem Mass Spectrometry (RRLC-ESI-MSn was used to separate and identify related substances in ampicillin capsule. The fragmentation behaviors of related substances were used to identify their chemical structures. Finally, a total of 13 related substances in ampicillin capsule were identified, including four identified components for the first time and three groups of isomers on the basis of the exact mass, fragmentation behaviors, retention time, and chemical structures in the literature. This study avoided time-consuming and complex chemosynthesis of related substances of ampicillin and the results could be useful for the quality control of ampicillin capsule to guarantee its safety in clinic. In the meantime, it provided a good example for the rapid identification of chemical structures of related substances of drugs.

  14. Rapid identification and simultaneous analysis of multiple constituents from Rheum tanguticum Maxim. ex Balf. by UPLC/Q-TOF-MS.

    Science.gov (United States)

    Gao, Liang-Liang; Guo, Tao; Xu, Xu-Dong; Yang, Jun-Shan

    2017-07-01

    Rhubarb contains biologically active compounds such as anthraquinones, anthrones, stilbenes and tannins. A rapid and efficient UPLC/Q-TOF-MS/MS method was developed and applied towards identifying the constituents of Rheum tanguticum Maxim. ex Balf. for the first time. Chemical constituents were separated and investigated by UPLC/Q-TOF-MS/MS in the negative ion mode. The ESI-MS 2 fragmentation pathways of four types of compounds were interpreted, providing a very useful guidance for the characterisation of different types of compounds. Based on the exact mass information, fragmentation characteristic and LC retention time of 7 reference standards, 30 constituents were tentatively identified from the methanol extract of R. tanguticum. Among them, seven compounds were described for the first time from R. tanguticum and two from the genus Rheum were described for the first time. The analytical tool used here is valuable for the rapid separation and identification of multiple and minor constituents in methanol extracts of R. tanguticum.

  15. Near Infrared Spectroscopy Facilitates Rapid Identification of Both Young and Mature Amazonian Tree Species.

    Science.gov (United States)

    Lang, Carla; Costa, Flávia Regina Capellotto; Camargo, José Luís Campana; Durgante, Flávia Machado; Vicentini, Alberto

    2015-01-01

    Precise identification of plant species requires a high level of knowledge by taxonomists and presence of reproductive material. This represents a major limitation for those working with seedlings and juveniles, which differ morphologically from adults and do not bear reproductive structures. Near-infrared spectroscopy (FT-NIR) has previously been shown to be effective in species discrimination of adult plants, so if young and adults have a similar spectral signature, discriminant functions based on FT-NIR spectra of adults can be used to identify leaves from young plants. We tested this with a sample of 419 plants in 13 Amazonian species from the genera Protium and Crepidospermum (Burseraceae). We obtained 12 spectral readings per plant, from adaxial and abaxial surfaces of dried leaves, and compared the rate of correct predictions of species with discriminant functions for different combinations of readings. We showed that the best models for predicting species in early developmental stages are those containing spectral data from both young and adult plants (98% correct predictions of external samples), but even using only adult spectra it is still possible to attain good levels of identification of young. We obtained an average of 75% correct identifications of young plants by discriminant equations based only on adults, when the most informative wavelengths were selected. Most species were accurately predicted (75-100% correct identifications), and only three had poor predictions (27-60%). These results were obtained despite the fact that spectra of young individuals were distinct from those of adults when species were analyzed individually. We concluded that FT-NIR has a high potential in the identification of species even at different ontogenetic stages, and that young plants can be identified based on spectra of adults with reasonable confidence.

  16. Mutations of NPM1 gene in de novo acute myeloid leukaemia: determination of incidence, distribution pattern and identification of two novel mutations in Indian population.

    Science.gov (United States)

    Ahmad, Firoz; Mandava, Swarna; Das, Bibhu Ranjan

    2009-06-01

    Mutations in the nucleophosmin (NPM1) gene have been recently described to occur in about one-third of acute myeloid leukaemias (AMLs) and represent the most frequent genetic alteration currently known in this subset, specially in those with normal karyotype. This study explored the prevalence and clinical profile of NPM1 mutations in a cohort of 200 Indian adult and children with AML. NPM1 mutations were observed in 19.5% of all population and 34.2% of those with normal karyotype. Adults had a significantly higher incidence of NPM1 mutations than children [38 of 161 (23.6%) vs. 1 of 39 (2.5%), p = 0.002]. NPM1 mutations were significantly associated with normal karyotype (p = 0.001), high WBC count (p = 0.034), AML-M4 subtype (p = 0.039) and a gradient increase of mutation rate with the increase in age groups. Sequence analysis of 39 mutated cases revealed typical mutations (types A, B, D, Nm and H*) as well as two novel variations (types F1 and F2). Majority of the patients had mutation type A (69.2%), followed by B (5.1%), D (15.3%), H* (2.5%) and Nm (2.5%) all involving COOH terminal of the NPM1 protein. In conclusion, this study represents the first report of NPM1 mutation from Indian population and confirms that the incidence of NPM1 mutations varies considerably globally, with slightly lower incidence in Indian population compared to western countries. The current study also served to identify two novel NPM1 mutants that add new insights into the heterogeneity of genomic insertions at exon 12. More ongoing larger studies are warranted to elucidate the molecular pathogenesis of AML that arises in this part of the world. Furthermore, we believe that in light of its high prevalence worldwide, inclusion of NPM1 mutation detection assay in diagnostic evaluations of AML may improve the efficacy of routine genetic characterization and allow assignment of patients to better-defined risk categories.

  17. Identification of Staphylococcus species and subspecies with the MicroScan Pos ID and Rapid Pos ID panel systems.

    Science.gov (United States)

    Kloos, W E; George, C G

    1991-01-01

    The accuracies of the MicroScan Pos ID and Rapid Pos ID panel systems (Baxter Diagnostic Inc., MicroScan Division, West Sacramento, Calif.) were compared with each other and with the accuracies of conventional methods for the identification of 25 Staphylococcus species and 4 subspecies. Conventional methods included those used in the original descriptions of species and subspecies and DNA-DNA hybridization. The Pos ID panel uses a battery of 18 tests, and the Rapid Pos ID panel uses a battery of 42 tests for the identification of Staphylococcus species. The Pos ID panel has modified conventional and chromogenic tests that can be read after 15 to 48 h of incubation; the Rapid Pos ID panel has tests that use fluorogenic substrates or fluorometric indicators, and test results can be read after 2 h of incubation in the autoSCAN-W/A. Results indicated that both MicroScan systems had a high degree of congruence (greater than or equal to 90%) with conventional methods for the species S. capitis, S. aureus, S. auricularis, S. saprophyticus, S. cohnii, S. arlettae, S. carnosus, S. lentus, and S. sciuri and, in particular, the subspecies S. capitis subsp. capitis and S. cohnii subsp. cohnii. The Rapid Pos ID panel system also had greater than or equal to 90% congruence with conventional methods for S. epidermidis, S. caprae, S. warneri subsp. 2, S. xylosus, S. kloosii, and S. caseolyticus. For both MicroScan systems, congruence with conventional methods was 80 to 90% for S. haemolyticus subsp. 1, S. equorum, S. intermedius, and S. hyicus; and in addition, with the Rapid Pos ID panel system congruence was 80 to 89% for S. capitis subsp. ureolyticus, S. warneri subsp. 1, S. hominis, S. cohnii subsp. urealyticum, and S. simulans. The MicroScan systems identified a lower percentage (50 to 75%) of strains of S. lugdunensis, S. gallinarum, S. schleiferi, and S. chromogenes, although the addition of specific tests to the systems might increase the accuracy of identification

  18. Rapid and inexpensive body fluid identification by RNA profiling-based multiplex High Resolution Melt (HRM) analysis.

    Science.gov (United States)

    Hanson, Erin K; Ballantyne, Jack

    2013-01-01

    Positive identification of the nature of biological material present on evidentiary items can be crucial for understanding the circumstances surrounding a crime. However, traditional protein-based methods do not permit the identification of all body fluids and tissues, and thus molecular based strategies for the conclusive identification of all forensically relevant biological fluids and tissues need to be developed. Messenger RNA (mRNA) profiling is an example of such a molecular-based approach. Current mRNA body fluid identification assays involve capillary electrophoresis (CE) or quantitative RT-PCR (qRT-PCR) platforms, each with its own limitations. Both platforms require the use of expensive fluorescently labeled primers or probes. CE-based assays require separate amplification and detection steps thus increasing the analysis time. For qRT-PCR assays, only 3-4 markers can be included in a single reaction since each requires a different fluorescent dye. To simplify mRNA profiling assays, and reduce the time and cost of analysis, we have developed single- and multiplex body fluid High Resolution Melt (HRM) assays for the identification of common forensically relevant biological fluids and tissues. The incorporated biomarkers include IL19 (vaginal secretions), IL1F7 (skin), ALAS2 (blood), MMP10 (menstrual blood), HTN3 (saliva) and TGM4 (semen).  The HRM assays require only unlabeled PCR primers and a single saturating intercalating fluorescent dye (Eva Green). Each body-fluid-specific marker can easily be identified by the presence of a distinct melt peak. Usually, HRM assays are used to detect variants or isoforms for a single gene target. However, we have uniquely developed duplex and triplex HRM assays to permit the simultaneous detection of multiple targets per reaction. Here we describe the development and initial performance evaluation of the developed HRM assays. The results demonstrate the potential use of HRM assays for rapid, and relatively inexpensive

  19. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry for rapid identification of fungal rhinosinusitis pathogens.

    Science.gov (United States)

    Huang, Yanfei; Wang, Jinglin; Zhang, Mingxin; Zhu, Min; Wang, Mei; Sun, Yufeng; Gu, Haitong; Cao, Jingjing; Li, Xue; Zhang, Shaoya; Lu, Xinxin

    2017-03-01

    Filamentous fungi are among the most important pathogens, causing fungal rhinosinusitis (FRS). Current laboratory diagnosis of FRS pathogens mainly relies on phenotypic identification by culture and microscopic examination, which is time consuming and expertise dependent. Although matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS has been employed to identify various fungi, its efficacy in the identification of FRS fungi is less clear. A total of 153 FRS isolates obtained from patients were analysed at the Clinical Laboratory at the Beijing Tongren Hospital affiliated to the Capital Medical University, between January 2014 and December 2015. They were identified by traditional phenotypic methods and Bruker MALDI-TOF MS (Bruker, Biotyper version 3.1), respectively. Discrepancies between the two methods were further validated by sequencing. Among the 153 isolates, 151 had correct species identification using MALDI-TOF MS (Bruker, Biot 3.1, score ≥2.0 or 2.3). MALDI-TOF MS enabled identification of some very closely related species that were indistinguishable by conventional phenotypic methods, including 1/10 Aspergillus versicolor, 3/20 Aspergillus flavus, 2/30 Aspergillus fumigatus and 1/20 Aspergillus terreus, which were misidentified by conventional phenotypic methods as Aspergillus nidulans, Aspergillus oryzae, Aspergillus japonicus and Aspergillus nidulans, respectively. In addition, 2/2 Rhizopus oryzae and 1/1 Rhizopus stolonifer that were identified only to the genus level by the phenotypic method were correctly identified by MALDI-TOF MS. MALDI-TOF MS is a rapid and accurate technique, and could replace the conventional phenotypic method for routine identification of FRS fungi in clinical microbiology laboratories.

  20. DNA detection and single nucleotide mutation identification using SERS for molecular diagnostics and global health

    Science.gov (United States)

    Ngo, Hoan T.; Gandra, Naveen; Fales, Andrew M.; Taylor, Steve M.; Vo-Dinh, Tuan

    2017-02-01

    Nucleic acid-based molecular diagnostics at the point-of-care (POC) and in resource-limited settings is still a challenge. We present a sensitive yet simple DNA detection method with single nucleotide polymorphism (SNP) identification capability. The detection scheme involves sandwich hybridization of magnetic beads conjugated with capture probes, target sequences, and ultrabright surface-enhanced Raman Scattering (SERS) nanorattles conjugated with reporter probes. Upon hybridization, the sandwich probes are concentrated at the detection focus controlled by a magnetic system for SERS measurements. The ultrabright SERS nanorattles, consisting of a core and a shell with resonance Raman reporters loaded in the gap space between the core and the shell, serve as SERS tags for ultrasensitive signal detection. Specific DNA sequences of the malaria parasite Plasmodium falciparum and dengue virus 1 (DENV1) were used as the model marker system. Detection limit of approximately 100 attomoles was achieved. Single nucleotide polymorphism (SNP) discrimination of wild type malaria DNA and mutant malaria DNA, which confers resistance to artemisinin drugs, was also demonstrated. The results demonstrate the molecular diagnostic potential of the nanorattle-based method to both detect and genotype infectious pathogens. The method's simplicity makes it a suitable candidate for molecular diagnosis at the POC and in resource-limited settings.

  1. Identification of recently selected mutations driven by artificial selection in hanwoo (korean cattle).

    Science.gov (United States)

    Lim, Dajeong; Gondro, Cedric; Park, Hye Sun; Cho, Yong Min; Chai, Han Ha; Seong, Hwan Hoo; Yang, Bo Suk; Hong, Seong Koo; Chang, Won Kyung; Lee, Seung Hwan

    2013-05-01

    Hanwoo have been subjected over the last seventy years to intensive artificial selection with the aim of improving meat production traits such as marbling and carcass weight. In this study, we performed a signature of selection analysis to identify recent positive selected regions driven by a long-term artificial selection process called a breeding program using whole genome SNP data. In order to investigate homozygous regions across the genome, we estimated iES (integrated Extended Haplotype Homozygosity SNP) for the each SNPs. As a result, we identified two highly homozygous regions that seem to be strong and/or recent positive selection. Five genes (DPH5, OLFM3, S1PR1, LRRN1 and CRBN) were included in this region. To go further in the interpretation of the observed signatures of selection, we subsequently concentrated on the annotation of differentiated genes defined according to the iES value of SNPs localized close or within them. We also described the detection of the adaptive evolution at the molecular level for the genes of interest. As a result, this analysis also led to the identification of OLFM3 as having a strong signal of selection in bovine lineage. The results of this study indicate that artificial selection which might have targeted most of these genes was mainly oriented towards improvement of meat production.

  2. Identification of Recently Selected Mutations Driven by Artificial Selection in Hanwoo (Korean Cattle

    Directory of Open Access Journals (Sweden)

    Dajeong Lim

    2013-05-01

    Full Text Available Hanwoo have been subjected over the last seventy years to intensive artificial selection with the aim of improving meat production traits such as marbling and carcass weight. In this study, we performed a signature of selection analysis to identify recent positive selected regions driven by a long-term artificial selection process called a breeding program using whole genome SNP data. In order to investigate homozygous regions across the genome, we estimated iES (integrated Extended Haplotype Homozygosity SNP for the each SNPs. As a result, we identified two highly homozygous regions that seem to be strong and/or recent positive selection. Five genes (DPH5, OLFM3, S1PR1, LRRN1 and CRBN were included in this region. To go further in the interpretation of the observed signatures of selection, we subsequently concentrated on the annotation of differentiated genes defined according to the iES value of SNPs localized close or within them. We also described the detection of the adaptive evolution at the molecular level for the genes of interest. As a result, this analysis also led to the identification of OLFM3 as having a strong signal of selection in bovine lineage. The results of this study indicate that artificial selection which might have targeted most of these genes was mainly oriented towards improvement of meat production.

  3. The spectrum of HNF1A gene mutations in Greek patients with MODY3: relative frequency and identification of seven novel germline mutations.

    Science.gov (United States)

    Tatsi, Christina; Kanaka-Gantenbein, Christina; Vazeou-Gerassimidi, Adriani; Chrysis, Dionysios; Delis, Dimitrios; Tentolouris, Nikolaos; Dacou-Voutetakis, Catherine; Chrousos, George P; Sertedaki, Amalia

    2013-11-01

    Maturity-Onset Diabetes of the Young (MODY) is the most common type of monogenic diabetes accounting for 1-2% of the population with diabetes. The relative incidence of HNF1A-MODY (MODY3) is high in European countries; however, data are not available for the Greek population. The aims of this study were to determine the relative frequency of MODY3 in Greece, the type of the mutations observed, and their relation to the phenotype of the patients. Three hundred ninety-five patients were referred to our center because of suspected MODY during a period of 15 yr. The use of Denaturing Gradient Gel Electrophoresis of polymerase chain reaction amplified DNA revealed 72 patients carrying Glucokinase gene mutations (MODY2) and 8 patients carrying HNF1A gene mutations (MODY3). After using strict criteria, 54 patients were selected to be further evaluated by direct sequencing or by multiplex ligation probe amplification (MLPA) for the presence of HNF1A gene mutations. In 16 unrelated patients and 13 of their relatives, 15 mutations were identified in the HNF1A gene. Eight of these mutations were previously reported, whereas seven were novel. Clinical features, such as age of diabetes at diagnosis or severity of hyperglycemia, were not related to the mutation type or location. In our cohort of patients fulfilling strict clinical criteria for MODY, 12% carried an HNF1A gene mutation, suggesting that defects of this gene are responsible for a significant proportion of monogenic diabetes in the Greek population. No clear phenotype-genotype correlations were identified. © 2013 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

  4. Rapid and Accurate Molecular Identification of the Emerging Multidrug-Resistant Pathogen Candida auris.

    Science.gov (United States)

    Kordalewska, Milena; Zhao, Yanan; Lockhart, Shawn R; Chowdhary, Anuradha; Berrio, Indira; Perlin, David S

    2017-08-01

    Candida auris is an emerging multidrug-resistant fungal pathogen causing nosocomial and invasive infections associated with high mortality. C. auris is commonly misidentified as several different yeast species by commercially available phenotypic identification platforms. Thus, there is an urgent need for a reliable diagnostic method. In this paper, we present fast, robust, easy-to-perform and interpret PCR and real-time PCR assays to identify C. auris and related species: Candida duobushaemulonii , Candida haemulonii , and Candida lusitaniae Targeting rDNA region nucleotide sequences, primers specific for C. auris only or C. auris and related species were designed. A panel of 140 clinical fungal isolates was used in both PCR and real-time PCR assays followed by electrophoresis or melting temperature analysis, respectively. The identification results from the assays were 100% concordant with DNA sequencing results. These molecular assays overcome the deficiencies of existing phenotypic tests to identify C. auris and related species. Copyright © 2017 Kordalewska et al.

  5. MutAid: Sanger and NGS Based Integrated Pipeline for Mutation Identification, Validation and Annotation in Human Molecular Genetics.

    Directory of Open Access Journals (Sweden)

    Ram Vinay Pandey

    Full Text Available Traditional Sanger sequencing as well as Next-Generation Sequencing have been used for the identification of disease causing mutations in human molecular research. The majority of currently available tools are developed for research and explorative purposes and often do not provide a complete, efficient, one-stop solution. As the focus of currently developed tools is mainly on NGS data analysis, no integrative solution for the analysis of Sanger data is provided and consequently a one-stop solution to analyze reads from both sequencing platforms is not available. We have therefore developed a new pipeline called MutAid to analyze and interpret raw sequencing data produced by Sanger or several NGS sequencing platforms. It performs format conversion, base calling, quality trimming, filtering, read mapping, variant calling, variant annotation and analysis of Sanger and NGS data under a single platform. It is capable of analyzing reads from multiple patients in a single run to create a list of potential disease causing base substitutions as well as insertions and deletions. MutAid has been developed for expert and non-expert users and supports four sequencing platforms including Sanger, Illumina, 454 and Ion Torrent. Furthermore, for NGS data analysis, five read mappers including BWA, TMAP, Bowtie, Bowtie2 and GSNAP and four variant callers including GATK-HaplotypeCaller, SAMTOOLS, Freebayes and VarScan2 pipelines are supported. MutAid is freely available at https://sourceforge.net/projects/mutaid.

  6. MutAid: Sanger and NGS Based Integrated Pipeline for Mutation Identification, Validation and Annotation in Human Molecular Genetics.

    Science.gov (United States)

    Pandey, Ram Vinay; Pabinger, Stephan; Kriegner, Albert; Weinhäusel, Andreas

    2016-01-01

    Traditional Sanger sequencing as well as Next-Generation Sequencing have been used for the identification of disease causing mutations in human molecular research. The majority of currently available tools are developed for research and explorative purposes and often do not provide a complete, efficient, one-stop solution. As the focus of currently developed tools is mainly on NGS data analysis, no integrative solution for the analysis of Sanger data is provided and consequently a one-stop solution to analyze reads from both sequencing platforms is not available. We have therefore developed a new pipeline called MutAid to analyze and interpret raw sequencing data produced by Sanger or several NGS sequencing platforms. It performs format conversion, base calling, quality trimming, filtering, read mapping, variant calling, variant annotation and analysis of Sanger and NGS data under a single platform. It is capable of analyzing reads from multiple patients in a single run to create a list of potential disease causing base substitutions as well as insertions and deletions. MutAid has been developed for expert and non-expert users and supports four sequencing platforms including Sanger, Illumina, 454 and Ion Torrent. Furthermore, for NGS data analysis, five read mappers including BWA, TMAP, Bowtie, Bowtie2 and GSNAP and four variant callers including GATK-HaplotypeCaller, SAMTOOLS, Freebayes and VarScan2 pipelines are supported. MutAid is freely available at https://sourceforge.net/projects/mutaid.

  7. Electromigration techniques - rapid methods for the detection and identification of urinary tract pathogens

    Czech Academy of Sciences Publication Activity Database

    Růžička, F.; Holá, V.; Horká, Marie

    2004-01-01

    Roč. 10, Suppl. 3 (2004), s. 621-622 ISSN 1198-743X. [14th ECCMID. European Congress of Clinical Microbiology and Infectious Diseases /14./. Praha, 01.05.2004-04.05.2004] R&D Projects: GA AV ČR IAA4031302 Institutional research plan: CEZ:AV0Z4031919 Keywords : electromigration techniques * identification * pathogens Subject RIV: CB - Analytical Chemistry, Separation Impact factor: 2.361, year: 2004

  8. Identification of a new mutation in an Iranian family with hereditary multiple osteochondromas

    Directory of Open Access Journals (Sweden)

    Akbaroghli S

    2016-12-01

    individuals. This mutation alters tryptophan to a premature stop codon on amino acid position 412 (p.Trp412x.Conclusion: The outcome of this study has extended the genotypic spectrum of Iranian patients with HMO, revealing a way for improving detection and genetic counseling in carriers. Keywords: bony exostoses, exon 4 of EXT1, HMO, hereditary multiple exostoses, HME, c.1235 G>A, EXT1 and EXT2

  9. Development of loop-mediated isothermal amplification (LAMP assay for rapid and sensitive identification of ostrich meat.

    Directory of Open Access Journals (Sweden)

    Amir Abdulmawjood

    Full Text Available Animal species identification is one of the primary duties of official food control. Since ostrich meat is difficult to be differentiated macroscopically from beef, therefore new analytical methods are needed. To enforce labeling regulations for the authentication of ostrich meat, it might be of importance to develop and evaluate a rapid and reliable assay. In the present study, a loop-mediated isothermal amplification (LAMP assay based on the cytochrome b gene of the mitochondrial DNA of the species Struthio camelus was developed. The LAMP assay was used in combination with a real-time fluorometer. The developed system allowed the detection of 0.01% ostrich meat products. In parallel, a direct swab method without nucleic acid extraction using the HYPLEX LPTV buffer was also evaluated. This rapid processing method allowed detection of ostrich meat without major incubation steps. In summary, the LAMP assay had excellent sensitivity and specificity for detecting ostrich meat and could provide a sampling-to-result identification-time of 15 to 20 minutes.

  10. Rapid identification and quantitative analysis of chemical constituents of Gentiana veitchiorum by UHPLC-PDA-QTOF-MS

    Directory of Open Access Journals (Sweden)

    Shan Li

    Full Text Available ABSTRACT Gentiana veitchiorum Hemsl., Gentianaceae, a traditional Tibetan medicine, was used for the treatment of liver jaundice with damp-heat pathogen, as well as for headache and chronic pharyngitis. A rapid ultra-performance liquid chromatography, photodiode array detector, quadrupole time-of-flight mass spectrometry method was developed for the fast and accurate identification and quantification of the chemical constituents of G. veitchiorum. In fact, eighteen compounds were detected and identified on the basis of their mass spectra, fragment characteristics and comparison with published data. Especially, the MS fragmentation pathways of iridoid glycosides and flavone C-glycosides were illustrated. Five compounds among them were quantified by UHPLC-PDA, including swertiamarin, gentiopicroside, sweroside, isoorientin, and isovitexin. The proposed method was then validated based on the analyses of linearity, accuracy, precision, and recovery. The overall recoveries for the five analytes ranged from 96.54% to 100.81%, with RSD from 1.05% to 1.82%. In addition, ten batches of G. veitchiorum from different areas were also analyzed. The developed method was rapid and reliable for both identification and quantification of the chemical constituents of G. veitchiorum, especially for simultaneous qualitative and quantitative analysis of iridoid glycosides and flavone C-glycosides.

  11. Rapid identification of salmonella serotypes with stereo and hyperspectral microscope imaging Methods

    Science.gov (United States)

    The hyperspectral microscope imaging (HMI) method can reduce detection time within 8 hours including incubation process. The early and rapid detection with this method in conjunction with the high throughput capabilities makes HMI method a prime candidate for implementation for the food industry. Th...

  12. Identification of a breast cancer family double heterozygote for RAD51C and BRCA2 gene mutations

    DEFF Research Database (Denmark)

    Ahlborn, Lise B; Steffensen, Ane Y; Jønson, Lars

    2015-01-01

    for mutations in the RAD51C and BRCA2 genes. The RAD51C missense mutation p.Arg258His has previously been identified in a homozygous state in a patient with Fanconi anemia. This mutation is known to affect the DNA repair function of the RAD51C protein. The BRCA2 p.Leu3216Leu synonymous mutation has not been...

  13. Rapid and Accurate Identification by Real-Time PCR of Biotoxin-Producing Dinoflagellates from the Family Gymnodiniaceae

    Directory of Open Access Journals (Sweden)

    Kirsty F. Smith

    2014-03-01

    Full Text Available The identification of toxin-producing dinoflagellates for monitoring programmes and bio-compound discovery requires considerable taxonomic expertise. It can also be difficult to morphologically differentiate toxic and non-toxic species or strains. Various molecular methods have been used for dinoflagellate identification and detection, and this study describes the development of eight real-time polymerase chain reaction (PCR assays targeting the large subunit ribosomal RNA (LSU rRNA gene of species from the genera Gymnodinium, Karenia, Karlodinium, and Takayama. Assays proved to be highly specific and sensitive, and the assay for G. catenatum was further developed for quantification in response to a bloom in Manukau Harbour, New Zealand. The assay estimated cell densities from environmental samples as low as 0.07 cells per PCR reaction, which equated to three cells per litre. This assay not only enabled conclusive species identification but also detected the presence of cells below the limit of detection for light microscopy. This study demonstrates the usefulness of real-time PCR as a sensitive and rapid molecular technique for the detection and quantification of micro-algae from environmental samples.

  14. Rapid and accurate identification by real-time PCR of biotoxin-producing dinoflagellates from the family gymnodiniaceae.

    Science.gov (United States)

    Smith, Kirsty F; de Salas, Miguel; Adamson, Janet; Rhodes, Lesley L

    2014-03-07

    The identification of toxin-producing dinoflagellates for monitoring programmes and bio-compound discovery requires considerable taxonomic expertise. It can also be difficult to morphologically differentiate toxic and non-toxic species or strains. Various molecular methods have been used for dinoflagellate identification and detection, and this study describes the development of eight real-time polymerase chain reaction (PCR) assays targeting the large subunit ribosomal RNA (LSU rRNA) gene of species from the genera Gymnodinium, Karenia, Karlodinium, and Takayama. Assays proved to be highly specific and sensitive, and the assay for G. catenatum was further developed for quantification in response to a bloom in Manukau Harbour, New Zealand. The assay estimated cell densities from environmental samples as low as 0.07 cells per PCR reaction, which equated to three cells per litre. This assay not only enabled conclusive species identification but also detected the presence of cells below the limit of detection for light microscopy. This study demonstrates the usefulness of real-time PCR as a sensitive and rapid molecular technique for the detection and quantification of micro-algae from environmental samples.

  15. Rapid identification of moulds and arthroconidial yeasts from positive blood cultures by MALDI-TOF mass spectrometry.

    Science.gov (United States)

    de Almeida, João N; Sztajnbok, Jaques; da Silva, Afonso Rafael; Vieira, Vinicius Adriano; Galastri, Anne Layze; Bissoli, Leandro; Litvinov, Nadia; Del Negro, Gilda Maria Barbaro; Motta, Adriana Lopes; Rossi, Flávia; Benard, Gil

    2016-11-01

    Moulds and arthroconidial yeasts are potential life-threatening agents of fungemia in immunocompromised patients. Fast and accurate identification (ID) of these pathogens hastens initiation of targeted antifungal therapy, thereby improving the patients' prognosis. We describe a new strategy that enabled the identification of moulds and arthroconidial yeasts directly from positive blood cultures by MALDI-TOF mass spectrometry (MS). Positive blood cultures (BCs) with Gram staining showing hyphae and/or arthroconidia were prospectively selected and submitted to an in-house protein extraction protocol. Mass spectra were obtained by Vitek MS™ system, and identifications were carried out with in the research use only (RUO) mode with an extended database (SARAMIS™ [v.4.12] plus in-house database). Fusarium solani, Fusarium verticillioides, Exophiala dermatitidis, Saprochaete clavata, and Trichosporon asahii had correct species ID by MALDI-TOF MS analysis of positive BCs. All cases were related to critically ill patients with high mortality fungemia and direct ID from positive BCs was helpful for rapid administration of targeted antifungal therapy. © The Author 2016. Published by Oxford University Press on behalf of The International Society for Human and Animal Mycology. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

  16. Automatic and rapid identification of glycopeptides by nano-UPLC-LTQ-FT-MS and proteomic search engine.

    Science.gov (United States)

    Giménez, Estela; Gay, Marina; Vilaseca, Marta

    2017-01-30

    Here we demonstrate the potential of nano-UPLC-LTQ-FT-MS and the Byonic™ proteomic search engine for the separation, detection, and identification of N- and O-glycopeptide glycoforms in standard glycoproteins. The use of a BEH C18 nanoACQUITY column allowed the separation of the glycopeptides present in the glycoprotein digest and a baseline-resolution of the glycoforms of the same glycopeptide on the basis of the number of sialic acids. Moreover, we evaluated several acquisition strategies in order to improve the detection and characterization of glycopeptide glycoforms with the maximum number of identification percentages. The proposed strategy is simple to set up with the technology platforms commonly used in proteomic labs. The method allows the straightforward and rapid obtention of a general glycosylated map of a given protein, including glycosites and their corresponding glycosylated structures. The MS strategy selected in this work, based on a gas phase fractionation approach, led to 136 unique peptides from four standard proteins, which represented 78% of the total number of peptides identified. Moreover, the method does not require an extra glycopeptide enrichment step, thus preventing the bias that this step could cause towards certain glycopeptide species. Data are available via ProteomeXchange with identifier PXD003578. We propose a simple and high-throughput glycoproteomics-based methodology that allows the separation of glycopeptide glycoforms on the basis of the number of sialic acids, and their automatic and rapid identification without prior knowledge of protein glycosites or type and structure of the glycans. Copyright © 2016 Elsevier B.V. All rights reserved.

  17. Reads2Type: a web application for rapid microbial taxonomy identification

    DEFF Research Database (Denmark)

    Saputra, Dhany; Rasmussen, Simon; Larsen, Mette Voldby

    2015-01-01

    genome of microbial isolates. Therefore we have developed Reads2Type, a web-based tool for taxonomy identification based on whole bacterial genome sequence data. Raw sequencing data provided by the user are mapped against a set of marker probes that are derived from currently available bacteria complete......, as the entire computational analysis is done on the computer of whom utilizes the web application. This also prevents data privacy issues to arise. The Reads2Type tool is available at http://www.cbs.dtu.dk/~dhany/reads2type.html ....

  18. [Identification of a novel GPR143 mutation in a Chinese family affected with X-linked ocular albinism].

    Science.gov (United States)

    Zhao, Qi; Guan, Menglong; Wang, Ling; Liao, Yong; Li-Ling, Jesse; Wan, Huajing

    2017-04-10

    To detect mutation of GPR143 gene in a Chinese patient affected with ocular albinism. Peripheral blood samples were collected from the proband and his parents. The coding regions of the GPR143 gene were subjected to PCR amplification and Sanger sequencing. A previously unreported mutation (c.758T>A) was found in exon 6 of the GPR143 gene in the proband and his mother. The same mutation was not found in his father. As predicted, the mutation has resulted in a stop codon, causing premature termination of protein translation. A novel mutation of the GPR143 gene related to X-linked ocular albinism has been identified.

  19. A simple, rapid and inexpensive screening method for the identification of Pythium insidiosum.

    Science.gov (United States)

    Tondolo, Juliana Simoni Moraes; Loreto, Erico Silva; Denardi, Laura Bedin; Mario, Débora Alves Nunes; Alves, Sydney Hartz; Santurio, Janio Morais

    2013-04-01

    Growth of Pythium insidiosum mycelia around minocycline disks (30μg) did not occur within 7days of incubation at 35°C when the isolates were grown on Sabouraud, corn meal, Muller-Hinton or RPMI agar. This technique offers a simple and rapid method for the differentiation of P. insidiosum from true filamentous fungi. Copyright © 2013 Elsevier B.V. All rights reserved.

  20. Identification of Six Novel PTH1R Mutations in Families with a History of Primary Failure of Tooth Eruption

    DEFF Research Database (Denmark)

    Risom, Lotte; Christoffersen, Line Borck; Daugaard-Jensen, Jette

    2013-01-01

    Primary Failure of tooth Eruption (PFE) is a non-syndromic disorder which can be caused by mutations in the parathyroid hormone receptor 1 gene (PTH1R). Traditionally, the disorder has been identified clinically based on post-emergent failure of eruption of permanent molars. However, patients...... undergone surgical and/or orthodontic interventions, and identified novel PTH1R mutations in all. Four of the six mutations were predicted to abolish correct mRNA maturation either through introduction of premature stop codons (c.947C>A and c.1082G>A), or by altering correct mRNA splicing (c.544......-26_544-23del and c.989G>T). The latter was validated by transfection of minigenes. The six novel mutations expand the mutation spectrum for PFE from eight to 14 pathogenic mutations. Loss-of-function mutations in PTH1R are also associated with recessively inherited Blomstrand chondrodysplasia. We compiled all...

  1. Direct Analysis in Real-time Mass Spectrometry for Rapid Identification of Traditional Chinese Medicines with Coumarins as Primary Characteristics.

    Science.gov (United States)

    Chen, Zhiyong; Yang, Yuanyuan; Tao, Hongxun; Liao, Liping; Li, Ye; Zhang, Zijia

    2017-05-01

    The increasing popularity of traditional Chinese medicines (TCMs) necessitates rapid and reliable methods for controlling their quality. Direct analysis in real-time mass spectrometry (DART-MS) represents a novel approach to analysing TCMs. To develop a quick and reliable method of identifying TCMs with coumarins as primary characteristics. DART-MS coupled with ion trap mass spectrometry was employed to rapidly identify TCMs with coumarins as primary characteristics and to explore the ionisation mechanisms of simple coumarins, furocoumarins and pyranocoumarins in detail. With minimal sample pretreatment, mass spectra of Fraxini Cortex, Angelicae Pubescentis Radix, Peucedani Radix and Psoraleae Fructus samples were obtained within seconds. The operating parameters of the DART ion source (e.g. grid electrode voltage and ionisation gas temperature) were carefully investigated to obtain high-quality mass spectra. The mass spectra of samples and DART-MS/MS spectra of marker compounds were used to identify sample materials. Successful authentication was achieved by analysing the same materials of different origins. Some simple coumarins, furocoumarins and pyranocoumarins can be directly detected by DART-MS as marker compounds. Our results demonstrated that DART-MS can provide a rapid and reliable method for the identification of TCMs containing different configurations of coumarins; the method may also be applicable to other plants. Copyright © 2016 John Wiley & Sons, Ltd. Copyright © 2016 John Wiley & Sons, Ltd.

  2. Identification of pollutant sources in a rapidly developing urban river catchment in China

    Science.gov (United States)

    Huang, Jingshui; Yin, Hailong; Jomma, Seifeddine; Rode, Michael; Zhou, Qi

    2016-04-01

    Rapid economic development and urbanization worldwide cause serious ecological and environmental problems. A typical region that is in transition and requires systemic research for effective intervention is the rapidly developing city of Hefei in central P. R. China. In order to investigate the sources of pollutants over a one-year period in Nanfei River catchment that drains the city of Hefei, discharges were measured and water samples were taken and measured along the 14km river section at 10 sites for 4 times from 2013 to 2014. Overflow concentrations of combined sewer and separate storm drains were also measured by selecting 15 rain events in 4 typical drainage systems. Loads and budgets of water and different pollutant sources i.e., wastewater treatment plant (WWTP) effluent, urban drainage overflow, unknown wastewater were calculated. The water balance demonstrated that >70% of the discharge originated from WWTP effluent. Lack of clean upstream inflow thereby is threatening ecological safety and water quality. Furthermore, mass fluxes calculations revealed that >40% of the COD (Chemical Oxygen Demand) loads were from urban drainage overflow because of a large amount of discharge of untreated wastewater in pumping stations during rain events. WWTP effluent was the predominant source of the total nitrogen loads (>60%) and ammonia loads (>45%). However, the total phosphorous loads from three different sources are similar (˜1/3). Thus, our research provided a basis for appropriate and prior mitigation strategies (state-of-art of WWTP upgrade, sewer systems modification, storm water regulation and storage capacity improvement, etc.) for different precedence-controlled pollutants with the limited infrastructure investments in these rapidly developing urban regions.

  3. Rapid detection and identification of pathogenic mycobacteria by combining radiometric and nucleic acid probe methods

    International Nuclear Information System (INIS)

    Ellner, P.D.; Kiehn, T.E.; Cammarata, R.; Hosmer, M.

    1988-01-01

    The combination of radiometric methodology (BACTEC 12B) and probe technology for recovery and identification of mycobacteria was studied in two large hospital laboratories. The sediment from vials with positive growth indices was tested with DNA probes specific for Mycobacterium tuberculosis, Mycobacterium avium, and Mycobacterium intracellulare. The sensitivity of the radiometric method and the specificity of the probes resulted in a marked reduction in the time to the final report. Biochemical testing could be eliminated on isolates giving a positive reaction with one of the probes. Some 176 isolates of M. tuberculosis, 110 of M. avium, and 5 of M. intracellulare were recovered. Two-thirds of these isolates were detected and identified within 2 weeks of inoculation and the remainder was detected by 4 weeks, a reduction of 5 to 7 weeks to the final report

  4. A PCR-based strategy for simple and rapid identification of rough presumptive Salmonella isolates

    DEFF Research Database (Denmark)

    Hoorfar, Jeffrey; Baggesen, Dorte Lau; Porting, P.H.

    1999-01-01

    The purpose of the present study was to investigate the application of ready-to-go Salmonella PCR tests, based on dry chemistry, for final identification of rough presumptive Salmonella isolates. The results were compared with two different biotyping methods performed at two different laboratories......, which did not result in any DNA band. A total of 32 out of the 36 rough presumptive isolates were positive in the PCR. All but one isolate were also identified as Salmonella by the two biochemical methods. All 80 Salmonella strains were also tested in the two multiplex serogroup tests based on PCR beads....... The sensitivity of the BAX Salmonella PCR test was assessed by testing a total of 80 Salmonella isolates, covering most serogroups, which correctly identified all the Salmonella strains by resulting in one 800-bp band in the sample tubes. The specificity of the PCR was assessed using 20 non-Salmonella strains...

  5. Structural identification of short/middle span bridges by rapid impact testing: theory and verification

    International Nuclear Information System (INIS)

    Zhang, Jian; Wu, Z S; Zhang, Q Q; Guo, S L; Xu, D W

    2015-01-01

    A structural strain flexibility identification method by processing the multiple-reference impact testing data is proposed. First, a kind of novel long-gauge fiber optic sensor is developed for structural macro-strain monitoring. Second, the multiple-reference impact testing technology is employed, during which both the impacting force and structural strain responses are measured. The impact testing technology has unique merit because it is able to extract exact structural frequency response functions (FRFs), while other test methods, for instance ambient tests, can only output the FRFs with scaled magnitudes. Most importantly, the originality of the article is that a method of identifying the structural strain flexibility characteristic from the impact test data has been proposed, which is useful for structural static strain prediction and capacity evaluation. Examples of a six meter simple supported beam and a multiple-span continuous beam bridge have successfully verified the effectiveness of the proposed method. (paper)

  6. Structural identification of short/middle span bridges by rapid impact testing: theory and verification

    Science.gov (United States)

    Zhang, Jian; Zhang, Q. Q.; Guo, S. L.; Xu, D. W.; Wu, Z. S.

    2015-06-01

    A structural strain flexibility identification method by processing the multiple-reference impact testing data is proposed. First, a kind of novel long-gauge fiber optic sensor is developed for structural macro-strain monitoring. Second, the multiple-reference impact testing technology is employed, during which both the impacting force and structural strain responses are measured. The impact testing technology has unique merit because it is able to extract exact structural frequency response functions (FRFs), while other test methods, for instance ambient tests, can only output the FRFs with scaled magnitudes. Most importantly, the originality of the article is that a method of identifying the structural strain flexibility characteristic from the impact test data has been proposed, which is useful for structural static strain prediction and capacity evaluation. Examples of a six meter simple supported beam and a multiple-span continuous beam bridge have successfully verified the effectiveness of the proposed method.

  7. Extraction and identification of cyclobutanones from irradiated cheese employing a rapid direct solvent extraction method.

    Science.gov (United States)

    Tewfik, Ihab

    2008-01-01

    2-Alkylcyclobutanones (cyclobutanones) are accepted as chemical markers for irradiated foods containing lipid. However, current extraction procedures (Soxhlet-florisil chromatography) for the isolation of these markers involve a long and tedious clean-up regime prior to gas chromatography-mass spectrophotometry identification. This paper outlines an alternative isolation and clean-up method for the extraction of cyclobutanones in irradiated Camembert cheese. The newly developed direct solvent extraction method enables the efficient screening of large numbers of food samples and is not as resource intensive as the BS EN 1785:1997 method. Direct solvent extraction appears to be a simple, robust method and has the added advantage of a considerably shorter extraction time for the analysis of foods containing lipid.

  8. Identification and manipulation of the pleuromutilin gene cluster from Clitopilus passeckerianus for increased rapid antibiotic production

    Science.gov (United States)

    Bailey, Andy M.; Alberti, Fabrizio; Kilaru, Sreedhar; Collins, Catherine M.; de Mattos-Shipley, Kate; Hartley, Amanda J.; Hayes, Patrick; Griffin, Alison; Lazarus, Colin M.; Cox, Russell J.; Willis, Christine L.; O'Dwyer, Karen; Spence, David W.; Foster, Gary D.

    2016-05-01

    Semi-synthetic derivatives of the tricyclic diterpene antibiotic pleuromutilin from the basidiomycete Clitopilus passeckerianus are important in combatting bacterial infections in human and veterinary medicine. These compounds belong to the only new class of antibiotics for human applications, with novel mode of action and lack of cross-resistance, representing a class with great potential. Basidiomycete fungi, being dikaryotic, are not generally amenable to strain improvement. We report identification of the seven-gene pleuromutilin gene cluster and verify that using various targeted approaches aimed at increasing antibiotic production in C. passeckerianus, no improvement in yield was achieved. The seven-gene pleuromutilin cluster was reconstructed within Aspergillus oryzae giving production of pleuromutilin in an ascomycete, with a significant increase (2106%) in production. This is the first gene cluster from a basidiomycete to be successfully expressed in an ascomycete, and paves the way for the exploitation of a metabolically rich but traditionally overlooked group of fungi.

  9. Rapid identification of bacterial resistance to Ciprofloxacin using surface-enhanced Raman spectroscopy

    Science.gov (United States)

    Kastanos, Evdokia; Hadjigeorgiou, Katerina; Pitris, Costas

    2014-02-01

    Due to its effectiveness and broad coverage, Ciprofloxacin is the fifth most prescribed antibiotic in the US. As current methods of infection diagnosis and antibiotic sensitivity testing (i.e. an antibiogram) are very time consuming, physicians prescribe ciprofloxacin before obtaining antibiogram results. In order to avoid increasing resistance to the antibiotic, a method was developed to provide both a rapid diagnosis and the sensitivity to the antibiotic. Using Surface Enhanced Raman Spectroscopy, an antibiogram was obtained after exposing the bacteria to Ciprofloxacin for just two hours. Spectral analysis revealed clear separation between sensitive and resistant bacteria and could also offer some inside into the mechanisms of resistance.

  10. Rapid and specific identification of Brucella abortus using the loop-mediated isothermal amplification (LAMP) assay.

    Science.gov (United States)

    Kang, Sung-Il; Her, Moon; Kim, Ji-Yeon; Lee, Jin Ju; Lee, Kichan; Sung, So-Ra; Jung, Suk Chan

    2015-06-01

    A rapid and accurate diagnosis of brucellosis is required to reduce and prevent the spread of disease among animals and the risk of transfer to humans. In this study, a Brucella abortus-specific (Ba) LAMP assay was developed, that had six primers designed from the BruAb2_0168 region of chromosome I. The specificity of this LAMP assay was confirmed with Brucella reference strains, B. abortus vaccine strains, B. abortus isolates and phylogenetically or serologically related strains. The detection limit of target DNA was up to 20 fg/μl within 60 min. The sensitivity of the new LAMP assay was equal to or slightly higher than other PCR based assays. Moreover, this Ba-LAMP assay could specifically amplify all B. abortus biovars compared to previous PCR assays. To our knowledge, this is the first report of specific detection of B. abortus using a LAMP assay. The Ba-LAMP assay can offer a rapid, sensitive and accurate diagnosis of bovine brucellosis in the field. Copyright © 2015 Elsevier Ltd. All rights reserved.

  11. Rapid identification of illegal synthetic adulterants in herbal anti-diabetic medicines using near infrared spectroscopy

    Science.gov (United States)

    Feng, Yanchun; Lei, Deqing; Hu, Changqin

    We created a rapid detection procedure for identifying herbal medicines illegally adulterated with synthetic drugs using near infrared spectroscopy. This procedure includes a reverse correlation coefficient method (RCCM) and comparison of characteristic peaks. Moreover, we made improvements to the RCCM based on new strategies for threshold settings. Any tested herbal medicine must meet two criteria to be identified with our procedure as adulterated. First, the correlation coefficient between the tested sample and the reference must be greater than the RCCM threshold. Next, the NIR spectrum of the tested sample must contain the same characteristic peaks as the reference. In this study, four pure synthetic anti-diabetic drugs (i.e., metformin, gliclazide, glibenclamide and glimepiride), 174 batches of laboratory samples and 127 batches of herbal anti-diabetic medicines were used to construct and validate the procedure. The accuracy of this procedure was greater than 80%. Our data suggest that this protocol is a rapid screening tool to identify synthetic drug adulterants in herbal medicines on the market.

  12. Proteotyping for the rapid identification of influenza virus and other biopathogens.

    Science.gov (United States)

    Downard, Kevin M

    2013-11-21

    The influenza virus is one of the most deadly infectious agents known to man and has been responsible for the deaths of some hundred million lives throughout human history. The need to rapidly and reliably survey circulating virus strains down to the molecular level is ever present. This tutorial describes the development and application of a new proteotyping approach that harnesses the power of high resolution of mass spectrometry to characterise the influenza virus, and by extension other bacterial and viral pathogens. The approach is shown to be able to type, subtype, and determine the lineage of human influenza virus strains through the detection of one or more signature peptide ions in the mass spectrum of whole virus digests. Pandemic strains can be similarly distinguished from seasonal ones, and new computer algorithms have been written to allow reassorted strains that pose the greatest pandemic risk to be rapidly identified from such datasets. The broader application of the approach is further demonstrated here for the parainfluenza virus, a virus which can be life threatening to children and presents similar clinical symptoms to influenza.

  13. Identification of a novel frameshift mutation in the ILDR1 gene in a UAE family, mutations review and phenotype genotype correlation.

    Directory of Open Access Journals (Sweden)

    Abdelaziz Tlili

    Full Text Available Autosomal recessive non-syndromic hearing loss is one of the most common monogenic diseases. It is characterized by high allelic and locus heterogeneities that make a precise diagnosis difficult. In this study, whole-exome sequencing was performed for an affected patient allowing us to identify a new frameshift mutation (c.804delG in the Immunoglobulin-Like Domain containing Receptor-1 (ILDR1 gene. Direct Sanger sequencing and segregation analysis were performed for the family pedigree. The mutation was homozygous in all affected siblings but heterozygous in the normal consanguineous parents. The present study reports a first ILDR1 gene mutation in the UAE population and confirms that the whole-exome sequencing approach is a robust tool for the diagnosis of monogenic diseases with high levels of allelic and locus heterogeneity. In addition, by reviewing all reported ILDR1 mutations, we attempt to establish a genotype phenotype correlation to explain the phenotypic variability observed at low frequencies.

  14. Case Report Identification of a novel SLC45A2 mutation in albinism by targeted next-generation sequencing.

    Science.gov (United States)

    Xue, J J; Xue, J F; Xue, H Q; Guo, Y Y; Liu, Y; Ouyang, N

    2016-09-19

    Albinism is a diverse group of hypopigmentary disorders caused by multiple-genetic defects. The genetic diagnosis of patients affected with albinism by Sanger sequencing is often complex, expensive, and time-consuming. In this study, we performed targeted next-generation sequencing to screen for 16 genes in a patient with albinism, and identified 21 genetic variants, including 19 known single nucleotide polymorphisms, one novel missense mutation (c.1456 G>A), and one disease-causing mutation (c.478 G>C). The novel mutation was not observed in 100 controls, and was predicted to be a damaging mutation by SIFT and Polyphen. Thus, we identified a novel mutation in SLC45A2 in a Chinese family, expanding the mutational spectrum of albinism. Our results also demonstrate that targeted next-generation sequencing is an effective genetic test for albinism.

  15. Identification of a novel FAM83H mutation and microhardness of an affected molar in autosomal dominant hypocalcified amelogenesis imperfecta.

    Science.gov (United States)

    Hyun, H-K; Lee, S-K; Lee, K-E; Kang, H-Y; Kim, E-J; Choung, P-H; Kim, J-W

    2009-11-01

    To determine the underlying molecular genetic aetiology of a family with the hypocalcified form of amelogenesis imperfecta and to investigate the hardness of the enamel and dentine of a known FAM83H mutation. Mutational screening of the FAM83H on the basis of candidate gene approach was performed. All exons and exon-intron boundaries was amplified and sequenced. A microhardness test was performed to measure the Vickers microhardness value. A novel nonsense mutation (c.1354C>T, p.Q452X) was identified in the last exon of FAM83H, which resulted in soft, uncalcified enamel. The affected enamel was extremely soft (about 17% of the normal control), but the underlying dentine was as hard as the normal control. Mutational analysis revealed a novel mutation in FAM83H gene. Hardness of dentine was not affected by the mutation, whilst the enamel was extremely soft.

  16. Rapid Evolution to Blast Crisis Associated with a Q252H ABL1 Kinase Domain Mutation in e19a2 BCR-ABL1 Chronic Myeloid Leukaemia

    Directory of Open Access Journals (Sweden)

    Sarah L. McCarron

    2013-01-01

    Full Text Available A minority of chronic myeloid leukaemia (CML patients express variant transcripts of which the e19a2 BCR-ABL1 fusion is the most common. Instances of tyrosine kinase inhibitor (TKI resistance in e19a2 BCR-ABL1 CML patients have rarely been reported. A case of e19a2 BCR-ABL1 CML is described in whom imatinib resistance, associated with a Q252H ABL1 kinase domain mutation, became apparent soon after initiation of TKI therapy. The patient rapidly transformed to myeloid blast crisis (BC with considerable bone marrow fibrosis and no significant molecular response to a second generation TKI. The clinical course was complicated by comorbidities with the patient rapidly succumbing to advanced disease. This scenario of Q252H-associated TKI resistance with rapid BC transformation has not been previously documented in e19a2 BCR-ABL1 CML. This case highlights the considerable challenges remaining in the management of TKI-resistant BC CML, particularly in the elderly patient.

  17. Evaluation and identification of factors related to KRAS and BRAF gene mutations in colorectal cancer: A meta-analysis

    Directory of Open Access Journals (Sweden)

    Li Lin

    2016-01-01

    Conclusion: The meta-analysis reveals that KRAS has a slightly higher mutation rate in MSI-L/MSS tumors. Moreover, BRAF mutations have higher detection rates in right-sided colorectal cancer, which suggests that BRAF mutations are likely in CIMP-H tumors. Therefore, based on these findings, the molecular diagnostic tests to be conducted in colorectal cancer patients can be determined according to the location/clinical features of the tumor.

  18. Rapid authentication and identification of different types of A. roxburghii by Tri-step FT-IR spectroscopy

    Science.gov (United States)

    Chen, Ying; Huang, Jinfang; Yeap, Zhao Qin; Zhang, Xue; Wu, Shuisheng; Ng, Chiew Hoong; Yam, Mun Fei

    2018-06-01

    Anoectochilus roxburghii (Wall.) Lindl. (Orchidaceae) is a precious traditional Chinese medicinal herb and has been perennially used to treat various illness. However, there were unethical sellers who adulterated wild A. roxburghii with tissue cultured and cultivated ones. Therefore, there is an urgent need for an effective authentication method to differentiate between these different types of A. roxburghii. In this research, the infrared spectroscopic tri-step identification approach including Fourier transform infrared spectroscopy (FT-IR), Second derivative infrared spectra (SD-IR) and two-dimensional correlation infrared spectra (2D-IR) was used to develop a simple and rapid method to discriminate between wild, cultivated and tissue cultivated A. roxburghii plant. Through this study, all three types of A. roxburghii plant were successfully identified and discriminated through the infrared spectroscopic tri-step identification method. Besides that, all the samples of wild, cultivated and tissue cultivated A. roxburghii plant were analysed with the Soft Independent Modelling of Class Analogy (SIMCA) pattern recognition technique to test and verify the experimental results. The results showed that the three types of A. roxburghii can be discriminated clearly as the recognition rate was 100% for all three types and the rejection rate was more than 60%. 70% of the validated samples were also identified correctly by the SIMCA model. The SIMCA model was also validated by comparing 70 standard herbs to the model. As a result, it was demonstrated that the macroscopic IR fingerprint method and the classification analysis could discriminate not only between the A. roxburghi samples and the standard herbs, it could also distinguish between the three different types of A. roxburghi plant in a direct, rapid and holistic manner.

  19. [Rapid Identification of Epicarpium Citri Grandis via Infrared Spectroscopy and Fluorescence Spectrum Imaging Technology Combined with Neural Network].

    Science.gov (United States)

    Pan, Sha-sha; Huang, Fu-rong; Xiao, Chi; Xian, Rui-yi; Ma, Zhi-guo

    2015-10-01

    To explore rapid reliable methods for detection of Epicarpium citri grandis (ECG), the experiment using Fourier Transform Attenuated Total Reflection Infrared Spectroscopy (FTIR/ATR) and Fluorescence Spectrum Imaging Technology combined with Multilayer Perceptron (MLP) Neural Network pattern recognition, for the identification of ECG, and the two methods are compared. Infrared spectra and fluorescence spectral images of 118 samples, 81 ECG and 37 other kinds of ECG, are collected. According to the differences in tspectrum, the spectra data in the 550-1 800 cm(-1) wavenumber range and 400-720 nm wavelength are regarded as the study objects of discriminant analysis. Then principal component analysis (PCA) is applied to reduce the dimension of spectroscopic data of ECG and MLP Neural Network is used in combination to classify them. During the experiment were compared the effects of different methods of data preprocessing on the model: multiplicative scatter correction (MSC), standard normal variable correction (SNV), first-order derivative(FD), second-order derivative(SD) and Savitzky-Golay (SG). The results showed that: after the infrared spectra data via the Savitzky-Golay (SG) pretreatment through the MLP Neural Network with the hidden layer function as sigmoid, we can get the best discrimination of ECG, the correct percent of training set and testing set are both 100%. Using fluorescence spectral imaging technology, corrected by the multiple scattering (MSC) results in the pretreatment is the most ideal. After data preprocessing, the three layers of the MLP Neural Network of the hidden layer function as sigmoid function can get 100% correct percent of training set and 96.7% correct percent of testing set. It was shown that the FTIR/ATR and fluorescent spectral imaging technology combined with MLP Neural Network can be used for the identification study of ECG and has the advantages of rapid, reliable effect.

  20. Rapid identification of genes controlling virulence and immunity in malaria parasites

    KAUST Repository

    Abkallo, Hussein M.

    2017-07-13

    Identifying the genetic determinants of phenotypes that impact disease severity is of fundamental importance for the design of new interventions against malaria. Here we present a rapid genome-wide approach capable of identifying multiple genetic drivers of medically relevant phenotypes within malaria parasites via a single experiment at single gene or allele resolution. In a proof of principle study, we found that a previously undescribed single nucleotide polymorphism in the binding domain of the erythrocyte binding like protein (EBL) conferred a dramatic change in red blood cell invasion in mutant rodent malaria parasites Plasmodium yoelii. In the same experiment, we implicated merozoite surface protein 1 (MSP1) and other polymorphic proteins, as the major targets of strain-specific immunity. Using allelic replacement, we provide functional validation of the substitution in the EBL gene controlling the growth rate in the blood stages of the parasites.

  1. Utilizing web-based geodata for rapid disaster identification and assessment

    Science.gov (United States)

    Leith, Kerry; Schmitt, Michael; Sickert, Salomon; Metzger, Alex; Krautblatter, Michael

    2014-05-01

    Developing methods to rapidly locate and quantify the impact of natural disasters can aid both the coordination of emergency response, and the long-term understanding of natural hazards in a range of environmental settings. Gaining such quantitative data in the aftermath of landslide events is particularly challenging, as the localized nature, steep terrain, and frequent damage to infrastructure caused by common triggering events (e.g. earthquakes, storms) complicates traditional methods of survey and data communication. As a result, the first, and often best overview of disastrous events is typically provided by eyewitness or first-responder photographs distributed through official, or social media networks. Although these images allow for an initial qualitative assessment of the event, their ad-hoc nature does not currently allow for either precise location, or quantitative evaluation of key event parameters (e.g. structural setting, geology, geometry, size, transport path, or total fall height). Here we present two tools designed to facilitate initial location and assessment of key event parameters using a combination of freely available geodata and information derived from eyewitness observations. These tools are currently under development, and rely on the adaptation of existing photogrammetric techniques in order to allow users to rapidly map and quantify event parameters from a combination of ad-hoc media photographs, and existing orthophoto and digital terrain model data (e.g. LiDAR, SRTM, ASTER). By incorporating results in freely-available GIS platforms such as Google Earth, local authorities will be able to to better assess and disseminate information regarding the impact of natural disasters in the critical hours following an event. We expect that quantitative data derived from events will provide important information to allow geohazard researchers to better assess landslide generation, and authorities to better plan responses to future triggering

  2. Identification of novel FBN1 and TGFBR2 mutations in 65 probands with Marfan syndrome or Marfan-like phenotypes.

    Science.gov (United States)

    Chung, Brian Hon-Yin; Lam, Stephen Tak-Sum; Tong, Tony Ming-For; Li, Susanna Yuk-Han; Lun, Kin-Shing; Chan, Daniel Hon-Chuen; Fok, Susanna Fung-Shan; Or, June Siu-Fong; Smith, David Keith; Yang, Wanling; Lau, Yu-Lung

    2009-07-01

    Marfan syndrome is an autosomal dominant connective tissue disorder, and mutations in the FBN1 and TGFBR2 genes have been identified in probands with MFS and related phenotypes. Using DHPLC and sequencing, we studied the mutation spectrum in 65 probands with Marfan syndrome and related phenotypes. A total of 24 mutations in FBN1 were identified, of which 19 (nine missense, six frameshift, two nonsense and two affecting splice junctions) were novel. In the remaining 41 probands, six were identified to have novel TGFBR2 mutations (one frameshift and five missense mutations). All novel mutations found in this study were confirmed to be absent in 50 unrelated normal individuals of the same ethnic background. In probands who fulfilled the Ghent criteria (n = 16), mutations in FBN1 were found in 81% of cases. None of those with TGFBR2 mutations fulfilled the Ghent criteria. Novel missense mutations of unknown significance were classified according to the latest ACMG guidelines and their likelihood to be causative was evaluated.

  3. Identification of two HEXA mutations causing infantile-onset Tay-Sachs disease in the Persian population.

    Science.gov (United States)

    Haghighi, Alireza; Rezazadeh, Jamileh; Shadmehri, Azam Ahmadi; Haghighi, Amirreza; Kornreich, Ruth; Desnick, Robert J

    2011-09-01

    The β-hexosaminidase A (HEXA) mutations in the first reported cases of infantile Tay-Sachs disease in the Persian population were identified in two unrelated consanguineous families. The clinical diagnoses of the affected infants were confirmed by their markedly deficient levels of HEXA activity in plasma or peripheral leukocytes. The specific causative mutation in each family was determined by sequencing the HEXA alleles in both sets of related parents. Two mutations were identified: c.1A>G (p.MIV), which obliterated the initiating methionine in codon 1, and c.1177C>T (p.R393X), which predicted a termination codon or nonsense mutation.

  4. Identification of somatic mutations in postmortem human brains by whole genome sequencing and their implications for psychiatric disorders.

    Science.gov (United States)

    Nishioka, Masaki; Bundo, Miki; Ueda, Junko; Katsuoka, Fumiki; Sato, Yukuto; Kuroki, Yoko; Ishii, Takao; Ukai, Wataru; Murayama, Shigeo; Hashimoto, Eri; Nagasaki, Masao; Yasuda, Jun; Kasai, Kiyoto; Kato, Tadafumi; Iwamoto, Kazuya

    2018-04-01

    Somatic mutations in the human brain are hypothesized to contribute to the functional diversity of brain cells as well as the pathophysiology of neuropsychiatric diseases. However, there are still few reports on somatic mutations in non-neoplastic human brain tissues. This study attempted to unveil the landscape of somatic mutations in the human brain. We explored the landscape of somatic mutations in human brain tissues derived from three individuals with no neuropsychiatric diseases by whole-genome deep sequencing at a depth of around 100. The candidate mutations underwent multi-layered filtering, and were validated by ultra-deep target amplicon sequencing at a depth of around 200 000. Thirty-one somatic mutations were identified in the human brain, demonstrating the utility of whole-genome sequencing of bulk brain tissue. The mutations were enriched in neuron-expressed genes, and two-thirds of the identified somatic single nucleotide variants in the brain tissues were cytosine-to-thymine transitions, half of which were in CpG dinucleotides. Our developed filtering and validation approaches will be useful to identify somatic mutations in the human brain. The vulnerability of neuron-expressed genes to mutational events suggests their potential relevance to neuropsychiatric diseases. © 2017 The Authors. Psychiatry and Clinical Neurosciences published by John Wiley & Sons Australia, Ltd on behalf of Japanese Society of Psychiatry and Neurology.

  5. Fluorescence excitation-emission matrix (EEM) spectroscopy for rapid identification and quality evaluation of cell culture media components.

    Science.gov (United States)

    Li, Boyan; Ryan, Paul W; Shanahan, Michael; Leister, Kirk J; Ryder, Alan G

    2011-11-01

    The application of fluorescence excitation-emission matrix (EEM) spectroscopy to the quantitative analysis of complex, aqueous solutions of cell culture media components was investigated. These components, yeastolate, phytone, recombinant human insulin, eRDF basal medium, and four different chemically defined (CD) media, are used for the formulation of basal and feed media employed in the production of recombinant proteins using a Chinese Hamster Ovary (CHO) cell based process. The comprehensive analysis (either identification or quality assessment) of these materials using chromatographic methods is time consuming and expensive and is not suitable for high-throughput quality control. The use of EEM in conjunction with multiway chemometric methods provided a rapid, nondestructive analytical method suitable for the screening of large numbers of samples. Here we used multiway robust principal component analysis (MROBPCA) in conjunction with n-way partial least squares discriminant analysis (NPLS-DA) to develop a robust routine for both the identification and quality evaluation of these important cell culture materials. These methods are applicable to a wide range of complex mixtures because they do not rely on any predetermined compositional or property information, thus making them potentially very useful for sample handling, tracking, and quality assessment in biopharmaceutical industries.

  6. Rapid Identification of Staphylococcus aureus Directly from Blood Cultures by Fluorescence In Situ Hybridization with Peptide Nucleic Acid Probes

    Science.gov (United States)

    Oliveira, Kenneth; Procop, Gary W.; Wilson, Deborah; Coull, James; Stender, Henrik

    2002-01-01

    A new fluorescence in situ hybridization (FISH) method with peptide nucleic acid (PNA) probes for identification of Staphylococcus aureus directly from positive blood culture bottles that contain gram-positive cocci in clusters (GPCC) is described. The test (the S. aureus PNA FISH assay) is based on a fluorescein-labeled PNA probe that targets a species-specific sequence of the 16S rRNA of S. aureus. Evaluations with 17 reference strains and 48 clinical isolates, including methicillin-resistant and methicillin-susceptible S. aureus species, coagulase-negative Staphylococcus species, and other clinically relevant and phylogenetically related bacteria and yeast species, showed that the assay had 100% sensitivity and 96% specificity. Clinical trials with 87 blood cultures positive for GPCC correctly identified 36 of 37 (97%) of the S. aureus-positive cultures identified by standard microbiological methods. The positive and negative predictive values were 100 and 98%, respectively. It is concluded that this rapid method (2.5 h) for identification of S. aureus directly from blood culture bottles that contain GPCC offers important information for optimal antibiotic therapy. PMID:11773123

  7. Multiplex-PCR As a Rapid and Sensitive Method for Identification of Meat Species in Halal-Meat Products.

    Science.gov (United States)

    Alikord, Mahsa; Keramat, Javad; Kadivar, Mahdi; Momtaz, Hassan; Eshtiaghi, Mohammad N; Homayouni-Rad, Aziz

    2017-01-01

    Species identification and authentication in meat products are important subjects for ensuring the health of consumers. The multiplex-PCR amplification and species- specific primer set were used for the identification of horse, donkey, pig and other ruminants in raw and processed meat products. Oligonucleotid primers were designed and patented for amplification of species-specific mitochondrial DNA sequences of each species and samples were prepared from binary meat mixtures. The results showed that meat species were accurately determined in all combinations by multiplex-PCR, and the sensitivity of this method was 0.001 ng, rendering this technique open to and suitable for use in industrial meat products. It is concluded that more fraud is seen in lower percentage industrial meat products than in higher percentage ones. There was also more fraud found in processed products than in raw ones. This rapid and useful test is recommended for quality control firms for applying more rigorous controls over industrial meat products, for the benefit of target consumers. Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.org.

  8. Rapid and reliable MALDI-TOF mass spectrometry identification of Candida non-albicans isolates from bloodstream infections.

    Science.gov (United States)

    Pulcrano, Giovanna; Iula, Dora Vita; Vollaro, Antonio; Tucci, Alessandra; Cerullo, Monica; Esposito, Matilde; Rossano, Fabio; Catania, Maria Rosaria

    2013-09-01

    Matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) fingerprinting has recently become an effective instrument for rapid microbiological diagnostics and in particular for identification of micro-organisms directly in a positive blood culture. The aim of the study was to evaluate a collection of 82 stored yeast isolates from bloodstream infection, by MALDI-TOF MS; 21 isolates were identified also directly from positive blood cultures and in the presence of other co-infecting micro-organisms. Of the 82 isolates grown on plates, 64 (76%) were correctly identified by the Vitek II system and 82 (100%) by MALDI-TOF MS; when the two methods gave different results, the isolate was identified by PCR. MALDI-TOF MS was unreliable in identifying two isolates (Candida glabrata and Candida parapsilosis) directly from blood culture; however, direct analysis from positive blood culture samples was fast and effective for the identification of yeast, which is of great importance for early and adequate treatment. © 2013. Published by Elsevier B.V. All rights reserved.

  9. 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.

  10. Rapid identification of dairy lactic acid bacteria by M13-generated, RAPD-PCR fingerprint databases.

    Science.gov (United States)

    Rossetti, Lia; Giraffa, Giorgio

    2005-11-01

    About a thousand lactic acid bacteria (LAB) isolated from dairy products, especially cheeses, were identified and typed by species-specific PCR and RAPD-PCR, respectively. RAPD-PCR profiles, which were obtained by using the M13 sequence as a primer, allowed us to implement a large database of different fingerprints, which were analysed by BioNumerics software. Cluster analysis of the combined RAPD-PCR fingerprinting profiles enabled us to implement a library, which is a collection of library units, which in turn is a selection of representative database entries. A library unit, in this case, can be considered to be a definable taxon. The strains belonged to 11 main RAPD-PCR fingerprinting library units identified as Lactobacillus casei/paracasei, Lactobacillus plantarum, Lactobacillus rhamnosus, Lactobacillus helveticus, Lactobacillus delbrueckii, Lactobacillus fermentum, Lactobacillus brevis, Enterococcus faecium, Enterococcus faecalis, Streptococcus thermophilus and Lactococcus lactis. The possibility to routinely identify newly typed, bacterial isolates by consulting the library of the software was valued. The proposed method could be suggested to refine previous strain identifications, eliminate redundancy and dispose of a technologically useful LAB strain collection. The same approach could also be applied to identify LAB strains isolated from other food ecosystems.

  11. Enumeration and rapid identification of yeasts during extraction processes of extra virgin olive oil in Tuscany.

    Science.gov (United States)

    Mari, Eleonora; Guerrini, Simona; Granchi, Lisa; Vincenzini, Massimo

    2016-06-01

    The aim of this study was to evaluate the occurrence of yeast populations during different olive oil extraction processes, carried out in three consecutive years in Tuscany (Italy), by analysing crushed pastes, kneaded pastes, oil from decanter and pomaces. The results showed yeast concentrations ranging between 10(3) and 10(5) CFU/g or per mL. Seventeen dominant yeast species were identified by random amplified polymorphic DNA with primer M13 and their identification was confirmed by restriction fragments length polymorphism of ribosomal internal transcribed spacer and sequencing rRNA genes. The isolation frequencies of each species in the collected samples pointed out that the occurrence of the various yeast species in olive oil extraction process was dependent not only on the yeasts contaminating the olives but also on the yeasts colonizing the plant for oil extraction. In fact, eleven dominant yeast species were detected from the washed olives, but only three of them were also found in oil samples at significant isolation frequency. On the contrary, the most abundant species in oil samples, Yamadazyma terventina, did not occur in washed olive samples. These findings suggest a phenomenon of contamination of the plant for oil extraction that selects some yeast species that could affect the quality of olive oil.

  12. Identification of Mutation of Glucose-6-Phosphate Dehy-drogenase (G6PD) in Iran: Meta- analysis Study.

    Science.gov (United States)

    Moosazadeh, Mahmood; Nekoei-Moghadam, Mahmood; Aliram-Zany, Maryam; Amiresmaili, Mohammadreza

    2013-09-01

    Glucose-6-phosphate dehydrogenase is one of the most common genetic deficiencies, which approximately 400 million people in the world suffer from. According to authors' initial search, numerous studies have been carried out in Iran regarding molecular variants of this enzyme. Thus, this meta-analysis presented a reliable estimation about prevalence of different types of molecular mutations of G6PD Enzyme in Iran. Keywords "glucose 6 phosphate dehydrogenase or G6PD, Mediterranean or Chatham or Cosenza and mutation, Iran or Iranian and their Persian equivalents" were searched in different databases. Moreover, reference list of the published studies were examined to increase sensitivity and to select more studies. After studying titles and abstracts of retrieved articles, excluding the repeated and unrelated ones, and evaluating quality of articles, documents were selected. Data was analyzed using STATA. After performing systematic review, 22 papers were entered this meta-analysis and 1698 subjects were examined concerning G6PD molecular mutation. In this meta-analysis, prevalence of Mediterranean mutation, Chatham mutation and Cosenza mutation in Iran was estimated 78.2%, 9.1% and 0.5% respectively. This meta-analysis showed that in spite of prevalence of different types of G6PD molecular mutations in center, north, north-west and west of Iran, the most common molecular mutations in people with G6PD deficiency in Iran, like other Mediterranean countries and countries around Persian Gulf, were Mediterranean mutation, Chatham mutation and Cosenza mutation. It is also recommended that future studies may focus on races and regions which haven't been taken into consideration up to now.

  13. The combined rapid detection and species-level identification of yeasts in simulated blood culture using a colorimetric sensor array.

    Science.gov (United States)

    Shrestha, Nabin K; Lim, Sung H; Wilson, Deborah A; SalasVargas, Ana Victoria; Churi, Yair S; Rhodes, Paul A; Mazzone, Peter J; Procop, Gary W

    2017-01-01

    A colorimetric sensor array (CSA) has been demonstrated to rapidly detect and identify bacteria growing in blood cultures by obtaining a species-specific "fingerprint" of the volatile organic compounds (VOCs) produced during growth. This capability has been demonstrated in prokaryotes, but has not been reported for eukaryotic cells growing in culture. The purpose of this study was to explore if a disposable CSA could differentially identify 7 species of pathogenic yeasts growing in blood culture. Culture trials of whole blood inoculated with a panel of clinically important pathogenic yeasts at four different microorganism loads were performed. Cultures were done in both standard BacT/Alert and CSA-embedded bottles, after adding 10 mL of spiked blood to each bottle. Color changes in the CSA were captured as images by an optical scanner at defined time intervals. The captured images were analyzed to identify the yeast species. Time to detection by the CSA was compared to that in the BacT/Alert system. One hundred sixty-two yeast culture trials were performed, including strains of several species of Candida (Ca. albicans, Ca. glabrata, Ca. parapsilosis, and Ca. tropicalis), Clavispora (synonym Candida) lusitaniae, Pichia kudriavzevii (synonym Candida krusei) and Cryptococcus neoformans, at loads of 8.2 × 105, 8.3 × 103, 8.5 × 101, and 1.7 CFU/mL. In addition, 8 negative trials (no yeast) were conducted. All negative trials were correctly identified as negative, and all positive trials were detected. Colorimetric responses were species-specific and did not vary by inoculum load over the 500000-fold range of loads tested, allowing for accurate species-level identification. The mean sensitivity for species-level identification by CSA was 74% at detection, and increased with time, reaching almost 95% at 4 hours after detection. At an inoculum load of 1.7 CFU/mL, mean time to detection with the CSA was 6.8 hours (17%) less than with the BacT/Alert platform. The CSA

  14. The combined rapid detection and species-level identification of yeasts in simulated blood culture using a colorimetric sensor array.

    Directory of Open Access Journals (Sweden)

    Nabin K Shrestha

    Full Text Available A colorimetric sensor array (CSA has been demonstrated to rapidly detect and identify bacteria growing in blood cultures by obtaining a species-specific "fingerprint" of the volatile organic compounds (VOCs produced during growth. This capability has been demonstrated in prokaryotes, but has not been reported for eukaryotic cells growing in culture. The purpose of this study was to explore if a disposable CSA could differentially identify 7 species of pathogenic yeasts growing in blood culture.Culture trials of whole blood inoculated with a panel of clinically important pathogenic yeasts at four different microorganism loads were performed. Cultures were done in both standard BacT/Alert and CSA-embedded bottles, after adding 10 mL of spiked blood to each bottle. Color changes in the CSA were captured as images by an optical scanner at defined time intervals. The captured images were analyzed to identify the yeast species. Time to detection by the CSA was compared to that in the BacT/Alert system.One hundred sixty-two yeast culture trials were performed, including strains of several species of Candida (Ca. albicans, Ca. glabrata, Ca. parapsilosis, and Ca. tropicalis, Clavispora (synonym Candida lusitaniae, Pichia kudriavzevii (synonym Candida krusei and Cryptococcus neoformans, at loads of 8.2 × 105, 8.3 × 103, 8.5 × 101, and 1.7 CFU/mL. In addition, 8 negative trials (no yeast were conducted. All negative trials were correctly identified as negative, and all positive trials were detected. Colorimetric responses were species-specific and did not vary by inoculum load over the 500000-fold range of loads tested, allowing for accurate species-level identification. The mean sensitivity for species-level identification by CSA was 74% at detection, and increased with time, reaching almost 95% at 4 hours after detection. At an inoculum load of 1.7 CFU/mL, mean time to detection with the CSA was 6.8 hours (17% less than with the BacT/Alert platform

  15. 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

  16. Evaluation of rapid immuno chromatographic assay kit using monoclonal mpt64 antibodies for identification of mycobacterium tuberculosis complex

    International Nuclear Information System (INIS)

    Satti, L.; Ikram, A.; Malik, N.

    2010-01-01

    To evaluate the performance of rapid immuno chromatographic kit MPT64 Ag for the identification of Mycobacterium tuberculosis complex from various Mycobacterium tuberculosis culture positive specimens. Department of Microbiology, Armed Forces Institute of Pathology Rawalpindi, from August 2008 through March 2009. Eighty four Mycobacterium tuberculosis positive cultures on I BACTEC 460 and MGIT 960, one ATCC 25177 MTB strain, three institutional control MTB strains, two institutional control MOTT strains and 20 different bacterial isolates were tested. Tests were performed according to the instructional manual. Out of total 84 tested samples, MPT64 showed positive result in 80 cultures. Only four positive cultures did not display any band on MPT64 kit. These four strains were reconfirmed as Mycobacterium tuberculosis by PCR method. MOTT control strains and all the 20 bacterial isolates were negative for band. The sensitivity and specificity of ICT assay in our study was 95.2% and 100% respectively. Rapid MPT64 Kit is a good diagnostic tool to differentiate between Mycobacterium tuberculosis complex and MOTT with 100% specificity. The technique is simple and can provide prompt information to the clinicians to initiate early and appropriate antituberculosis therapy. (author)

  17. Rapid and sensitive multiplex single-tube nested PCR for the identification of five human Plasmodium species.

    Science.gov (United States)

    Saito, Takahiro; Kikuchi, Aoi; Kaneko, Akira; Isozumi, Rie; Teramoto, Isao; Kimura, Masatsugu; Hirasawa, Noriyasu; Hiratsuka, Masahiro

    2018-06-01

    Malaria is caused by five species of Plasmodium in humans. Microscopy is currently used for pathogen detection, requiring considerable training and technical expertise as the parasites are often difficult to differentiate morphologically. Rapid diagnostic tests are as reliable as microscopy and offer faster diagnoses but possess lower detection limits and are incapable of distinguishing among the parasitic species. To improve global health efforts towards malaria control, a rapid, sensitive, species-specific, and economically viable diagnostic method is needed. In this study, we designed a malaria diagnostic method involving a multiplex single-tube nested PCR targeting Plasmodium mitochondrial cytochrome c oxidase III and single-stranded tag hybridization chromatographic printed-array strip. The detection sensitivity was found to be at least 40 times higher than that of agarose gel electrophoresis with ethidium bromide. This system also enables the identification of both single- and mixed-species malaria infections. The assay was validated with 152 Kenyan samples; using nested PCR as the standard, the assay's sensitivity and specificity were 88.7% and 100.0%, respectively. The turnaround time required, from PCR preparation to signal detection, is 90min. Our method should improve the diagnostic speed, treatment efficacy, and control of malaria, in addition to facilitating surveillance within global malaria eradication programs. Copyright © 2018 Elsevier B.V. All rights reserved.

  18. Rapid and reliable identification of Gram-negative bacteria and Gram-positive cocci by deposition of bacteria harvested from blood cultures onto the MALDI-TOF plate.

    Science.gov (United States)

    Barnini, Simona; Ghelardi, Emilia; Brucculeri, Veronica; Morici, Paola; Lupetti, Antonella

    2015-06-18

    Rapid identification of the causative agent(s) of bloodstream infections using the matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) methodology can lead to increased empirical antimicrobial therapy appropriateness. Herein, we aimed at establishing an easier and simpler method, further referred to as the direct method, using bacteria harvested by serum separator tubes from positive blood cultures and placed onto the polished steel target plate for rapid identification by MALDI-TOF. The results by the direct method were compared with those obtained by MALDI-TOF on bacteria isolated on solid media. Identification of Gram-negative bacilli was 100 % concordant using the direct method or MALDI-TOF on isolated bacteria (96 % with score > 2.0). These two methods were 90 % concordant on Gram-positive cocci (32 % with score > 2.0). Identification by the SepsiTyper method of Gram-positive cocci gave concordant results with MALDI-TOF on isolated bacteria in 87 % of cases (37 % with score > 2.0). The direct method herein developed allows rapid identification (within 30 min) of Gram-negative bacteria and Gram-positive cocci from positive blood cultures and can be used to rapidly report reliable and accurate results, without requiring skilled personnel or the use of expensive kits.

  19. Rapid identification of Chinese Sauce liquor from different fermentation positions with FT-IR spectroscopy

    Science.gov (United States)

    Li, Changwen; Wei, Jiping; Zhou, Qun; Sun, Suqin

    2008-07-01

    FT-IR and two-dimensional correlation spectroscopy (2D-IR) technology were applied to discriminate Chinese Sauce liquor from different fermentation positions (top, middle and bottom of fermentation cellar) for the first time. The liquors at top, middle and bottom of fermentation cellar, possessed the characteristic peaks at 1731 cm -1, 1733 cm -1 and 1602 cm -1, respectively. In the 2D correlation infrared spectra, the differences were amplified. A strong auto-peak at 1725 cm -1 showed in the 2D spectra of the Top Liquor, which indicated that the liquor might contain some ester compounds. Different from Top Liquor, three auto-peaks at 1695, 1590 and 1480 cm -1 were identified in 2D spectra of Middle Liquor, which were the characteristic absorption of acid, lactate. In 2D spectra of Bottom Liquor, two auto-peaks at 1570 and 1485 cm -1 indicated that lactate was the major component. As a result, FT-IR and 2D-IR correlation spectra technology provided a rapid and effective method for the quality analysis of the Sauce liquor.

  20. Development of a hybrid proximal sensing method for rapid identification of petroleum contaminated soils

    Energy Technology Data Exchange (ETDEWEB)

    Chakraborty, Somsubhra [Ramakrishna Mission Vivekananda University, Kolkata (India); Weindorf, David C., E-mail: david.weindorf@ttu.edu [Department of Plant and Soil Science, Texas Tech University, Lubbock, TX (United States); Li, Bin [Department of Experimental Statistics, Louisiana State University, Baton Rouge, LA (United States); Ali Aldabaa, Abdalsamad Abdalsatar [Desert Research Center, Cairo (Egypt); Ghosh, Rakesh Kumar [National Institute of Research on Jute and Allied Fibre Technology, Kolkata (India); Paul, Sathi; Nasim Ali, Md. [Ramakrishna Mission Vivekananda University, Kolkata (India)

    2015-05-01

    Using 108 petroleum contaminated soil samples, this pilot study proposed a new analytical approach of combining visible near-infrared diffuse reflectance spectroscopy (VisNIR DRS) and portable X-ray fluorescence spectrometry (PXRF) for rapid and improved quantification of soil petroleum contamination. Results indicated that an advanced fused model where VisNIR DRS spectra-based penalized spline regression (PSR) was used to predict total petroleum hydrocarbon followed by PXRF elemental data-based random forest regression was used to model the PSR residuals, it outperformed (R{sup 2} = 0.78, residual prediction deviation (RPD) = 2.19) all other models tested, even producing better generalization than using VisNIR DRS alone (RPD's of 1.64, 1.86, and 1.96 for random forest, penalized spline regression, and partial least squares regression, respectively). Additionally, unsupervised principal component analysis using the PXRF + VisNIR DRS system qualitatively separated contaminated soils from control samples. Capsule: Fusion of PXRF elemental data and VisNIR derivative spectra produced an optimized model for total petroleum hydrocarbon quantification in soils. - Highlights: • PXRF elemental data and VisNIR DRS spectra were correlated to soil TPH. • VisNIR + PSR was used to predict TPH followed by PXRF + RF to predict the residuals. • The fused model produced better results than other multivariate models tested.

  1. Rapid identification of the genus Dekkera/Brettanomyces, the Dekkera subgroup and all individual species.

    Science.gov (United States)

    Hulin, M; Harrison, E; Stratford, M; Wheals, A E

    2014-09-18

    The genus Dekkera/Brettanomyces comprises five described species: Dekkera bruxellensis, D. anomala, Brettanomyces custersianus, B. naardenensis and B. nanus. Some of them, especially D. bruxellensis, are important spoilage organisms, particularly in the wine and beverage industries. Because of their economic importance many different methods have been developed to identify members of the genus in general and D. bruxellensis in particular. These methods vary in their rapidity, complexity and cost but, partly because of confidentiality issues, it is unclear which methods are used, or how widely, in the relevant industries. Building on previous work with the genera Saccharomyces and Zygosaccharomyces, a suite of eight PCR primer pairs has been designed either on the D1-D2 region of the 26S rRNA gene or translation elongation factor TEF1-α. These primers can specifically identify the genus as a whole, only Dekkera species, each one of the five recognised species as well as a significant subgroup of D. bruxellensis represented by NCYC 3426. Multiplexing has also been tried and it has been shown to be possible with some combinations of genus or Dekkera-level and species-specific primers. Using direct colony PCR amplification followed by gel electrophoresis, a clear positive result can be obtained in less than 3h, thus providing a quick, reliable and inexpensive way to identify target species. Copyright © 2014 Elsevier B.V. All rights reserved.

  2. Rapid identification of vibrio-cholerae O1 by coaglutination test using mono-specifis antibody

    Directory of Open Access Journals (Sweden)

    Bazargan SA

    1996-07-01

    Full Text Available In our investigation, rabbit hyper-immune serum to V.cholerae ogawa was absorbed with V.cholerae inaba whole-cells and vice versa. Applying ammonium sulphate precipitation method, mono-specific g globulins were purified and concentrated from the absorbed whole serum. These antibodies were fixed on staphylococcus cowan 1 NCTC-8325 whole-cells, using different chemical fixatives. It was observed that maximum fixation of g globulin to protein-A was achieved by 1-propanol 50% at 3 hours, which revealed through single radial immuno-diffusion techniqe. The rectal swab samples were cultured in an enrichment bile-peptons broth. After 5 hours 37°C while agitations, one drop of each sample was mixed with one drop of vibrio-cholerae bivalent mono-specific coagglutination reagent (VBCR. The results were read after 2 to 3 minutes. Finally though statistical analysis sensitivity and specificity of coagglutination test were calculated to be 95.1% and 99.2% respectively, when compared to positive & negative controls and conventional culture methods. Using VBCR, coagglutination test can be therefore considered as a simple, reliable and rapid method to detect V.cholerae O1 in the stool of patients in endemic area and less equipped laboratories

  3. Rapid Classification and Identification of Microcystis aeruginosa Strains Using MALDI-TOF MS and Polygenetic Analysis.

    Directory of Open Access Journals (Sweden)

    Li-Wei Sun

    Full Text Available Matrix-assisted laser desorption-ionization-time-of-flight mass spectrometry (MALDI-TOF MS was used to establish a rapid, simple, and accurate method to differentiate among strains of Microcystis aeruginosa, one of the most prevalent types of bloom-forming cyanobacteria. M. aeruginosa NIES-843, for which a complete genome has been sequenced, was used to characterize ribosomal proteins as biomarkers and to optimize conditions for observing ribosomal proteins as major peaks in a given mass spectrum. Thirty-one of 52 ribosomal subunit proteins were detected and identified along the mass spectrum. Fifty-five strains of M. aeruginosa from different habitats were analyzed using MALDI-TOF MS; among these samples, different ribosomal protein types were observed. A polygenetic analysis was performed using an unweighted pair-group method with arithmetic means and different ribosomal protein types to classify the strains into five major clades. Two clades primarily contained toxic strains, and the other three clades contained exclusively non-toxic strains. This is the first study to differentiate cyanobacterial strains using MALDI-TOF MS.

  4. Rapid Classification and Identification of Microcystis aeruginosa Strains Using MALDI-TOF MS and Polygenetic Analysis.

    Science.gov (United States)

    Sun, Li-Wei; Jiang, Wen-Jing; Sato, Hiroaki; Kawachi, Masanobu; Lu, Xi-Wu

    2016-01-01

    Matrix-assisted laser desorption-ionization-time-of-flight mass spectrometry (MALDI-TOF MS) was used to establish a rapid, simple, and accurate method to differentiate among strains of Microcystis aeruginosa, one of the most prevalent types of bloom-forming cyanobacteria. M. aeruginosa NIES-843, for which a complete genome has been sequenced, was used to characterize ribosomal proteins as biomarkers and to optimize conditions for observing ribosomal proteins as major peaks in a given mass spectrum. Thirty-one of 52 ribosomal subunit proteins were detected and identified along the mass spectrum. Fifty-five strains of M. aeruginosa from different habitats were analyzed using MALDI-TOF MS; among these samples, different ribosomal protein types were observed. A polygenetic analysis was performed using an unweighted pair-group method with arithmetic means and different ribosomal protein types to classify the strains into five major clades. Two clades primarily contained toxic strains, and the other three clades contained exclusively non-toxic strains. This is the first study to differentiate cyanobacterial strains using MALDI-TOF MS.

  5. A rapid in vitro screening system for the identification and evaluation of anticancer drugs

    International Nuclear Information System (INIS)

    Kao, J.W.; Collins, J.L.

    1989-01-01

    We report the development of an in vitro screening system that can be used to identify new anticancer drugs that are specifically cytotoxic for dividing cells. The screening system takes advantage of the potential of many cell lines, including tumor cells, to stop dividing when they are plated at high cell density. The cytotoxic effects of anticancer drugs on dividing (i.e., cells plated at low cell density) and nondividing cells (i.e., cells plated at high cell density) is measured by the incorporation of 51Cr. This in vitro system was evaluated by measuring the cytotoxic effects of the anticancer drugs cisplatin, thiotepa, doxorubicin, methotrexate, and vinblastine on the cell lines B/C-N, ME-180, and MCF-7. In this in vitro system the concentrations of the anticancer drugs that produced significant cytotoxicity on only dividing cells are similar to the concentrations that are used clinically. The fact that this in vitro system is rapid, simple, applicable to many cell types, and able to predict effective concentrations of anticancer drugs should make it useful for the screening of new anticancer drugs and for the design of preclinical studies

  6. Rapid Identification of Potential Drugs for Diabetic Nephropathy Using Whole-Genome Expression Profiles of Glomeruli

    Directory of Open Access Journals (Sweden)

    Jingsong Shi

    2016-01-01

    Full Text Available Objective. To investigate potential drugs for diabetic nephropathy (DN using whole-genome expression profiles and the Connectivity Map (CMAP. Methodology. Eighteen Chinese Han DN patients and six normal controls were included in this study. Whole-genome expression profiles of microdissected glomeruli were measured using the Affymetrix human U133 plus 2.0 chip. Differentially expressed genes (DEGs between late stage and early stage DN samples and the CMAP database were used to identify potential drugs for DN using bioinformatics methods. Results. (1 A total of 1065 DEGs (FDR 1.5 were found in late stage DN patients compared with early stage DN patients. (2 Piperlongumine, 15d-PGJ2 (15-delta prostaglandin J2, vorinostat, and trichostatin A were predicted to be the most promising potential drugs for DN, acting as NF-κB inhibitors, histone deacetylase inhibitors (HDACIs, PI3K pathway inhibitors, or PPARγ agonists, respectively. Conclusion. Using whole-genome expression profiles and the CMAP database, we rapidly predicted potential DN drugs, and therapeutic potential was confirmed by previously published studies. Animal experiments and clinical trials are needed to confirm both the safety and efficacy of these drugs in the treatment of DN.

  7. Identification of two novel mutations in the PHEX gene in Chinese patients with hypophosphatemic rickets/osteomalacia.

    Science.gov (United States)

    Yue, Hua; Yu, Jin-bo; He, Jin-wei; Zhang, Zeng; Fu, Wen-zhen; Zhang, Hao; Wang, Chun; Hu, Wei-wei; Gu, Jie-mei; Hu, Yun-qiu; Li, Miao; Liu, Yu-juan; Zhang, Zhen-Lin

    2014-01-01

    X-linked dominant hypophosphatemia (XLH) is the most prevalent form of inherited rickets/osteomalacia in humans. The aim of this study was to identify PHEX gene mutations and describe the clinical features observed in 6 unrelated Chinese families and 3 sporadic patients with hypophosphatemic rickets/osteomalacia. For this study, 45 individuals from 9 unrelated families of Chinese Han ethnicity (including 16 patients and 29 normal phenotype subjects), and 250 healthy donors were recruited. All 22 exons and exon-intron boundaries of the PHEX gene were amplified by polymerase chain reaction (PCR) and directly sequenced. The PHEX mutations were detected in 6 familial and 3 sporadic hypophosphatemic rickets/osteomalacia. Altogether, 2 novel mutations were detected: 1 missense mutation c.1183G>C in exon 11, resulting in p.Gly395Arg and 1 missense mutation c.1751A>C in exon 17, resulting in p.His584Pro. No mutations were found in the 250 healthy controls. Our study increases knowledge of the PHEX gene mutation types and clinical phenotypes found in Chinese patients with XLH, which is important for understanding the genetic basis of XLH. The molecular diagnosis of a PHEX genetic mutation is of great importance for confirming the clinical diagnosis of XLH, conducting genetic counseling, and facilitating prenatal intervention, especially in the case of sporadic patients.

  8. Rapid evolution of troglomorphic characters suggests selection rather than neutral mutation as a driver of eye reduction in cave crabs.

    Science.gov (United States)

    Klaus, Sebastian; Mendoza, José C E; Liew, Jia Huan; Plath, Martin; Meier, Rudolf; Yeo, Darren C J

    2013-04-23

    This study asked whether reductive traits in cave organisms evolve at a slower pace (suggesting neutral evolution under relaxed selection) than constructive changes, which are likely to evolve under directional selection. We investigated 11 subterranean and seven surface populations of Sundathelphusa freshwater crabs on Bohol Island, Philippines, and examined constructive traits associated with improved food finding in darkness (increased leg and setae length) and reductive traits (reduced cornea size and eyestalk length). All changes occurred rapidly, given that the age of the most recent common ancestor was estimated to be 722-271 ka based on three mitochondrial markers. In order to quantify the speed of character change, we correlated the degree of morphological change with genetic distances between surface and subterranean individuals. The temporal pattern of character change following the transition to subterranean life was indistinguishable for constructive and reductive traits, characterized by an immediate onset and rapid evolutionary change. We propose that the evolution of these reductive traits-just like constructive traits-is most likely driven by strong directional selection.

  9. Identification of a novel mutation in WFS1 in a family affected by low-frequency hearing impairment

    Energy Technology Data Exchange (ETDEWEB)

    Kunz, Juergen; Marquez-Klaka, Ben; Uebe, Steffen; Volz-Peters, Anja; Berger, Roswitha; Rausch, Peter

    2003-04-09

    Previously we confirmed linkage of autosomal dominantly inherited low-frequency sensorineural hearing impairment (LFSNHI) in a German family to the genetic locus DFNA6/DFNA14 on chromosome 4p16.3 close to the markers D4S432 and D4S431. Analysis of data from the Human Genome Project, showed that WFS1 is located in this region. Mutations in WFS1 are known to be responsible for Wolfram syndrome (DIDMOAD, MIM no. 606201), which follows an autosomal recessive trait. Studies in low-frequency hearing loss families showed that mutations in WFS1 were responsible for the phenotype. In all affected family members analysed, we detected a missense mutation in WFS1 (K705N) and therefore confirm the finding that the majority of mutations responsible for LFSNHI are missense mutations which localise to the C-terminal domain of the protein.

  10. Identification of a novel mutation in WFS1 in a family affected by low-frequency hearing impairment

    International Nuclear Information System (INIS)

    Kunz, Juergen; Marquez-Klaka, Ben; Uebe, Steffen; Volz-Peters, Anja; Berger, Roswitha; Rausch, Peter

    2003-01-01

    Previously we confirmed linkage of autosomal dominantly inherited low-frequency sensorineural hearing impairment (LFSNHI) in a German family to the genetic locus DFNA6/DFNA14 on chromosome 4p16.3 close to the markers D4S432 and D4S431. Analysis of data from the Human Genome Project, showed that WFS1 is located in this region. Mutations in WFS1 are known to be responsible for Wolfram syndrome (DIDMOAD, MIM no. 606201), which follows an autosomal recessive trait. Studies in low-frequency hearing loss families showed that mutations in WFS1 were responsible for the phenotype. In all affected family members analysed, we detected a missense mutation in WFS1 (K705N) and therefore confirm the finding that the majority of mutations responsible for LFSNHI are missense mutations which localise to the C-terminal domain of the protein

  11. Identification of six novel PTH1R mutations in families with a history of primary failure of tooth eruption.

    Science.gov (United States)

    Risom, Lotte; Christoffersen, Line; Daugaard-Jensen, Jette; Hove, Hanne Dahlgaard; Andersen, Henriette Skovgaard; Andresen, Brage Storstein; Kreiborg, Sven; Duno, Morten

    2013-01-01

    Primary Failure of tooth Eruption (PFE) is a non-syndromic disorder which can be caused by mutations in the parathyroid hormone receptor 1 gene (PTH1R). Traditionally, the disorder has been identified clinically based on post-emergent failure of eruption of permanent molars. However, patients with PTH1R mutations will not benefit from surgical and/or orthodontic treatment and it is therefore clinically important to establish whether a given failure of tooth eruption is caused by a PTH1R defect or not. We analyzed the PTH1R gene in six patients clinically diagnosed with PFE, all of which had undergone surgical and/or orthodontic interventions, and identified novel PTH1R mutations in all. Four of the six mutations were predicted to abolish correct mRNA maturation either through introduction of premature stop codons (c.947C>A and c.1082G>A), or by altering correct mRNA splicing (c.544-26_544-23del and c.989G>T). The latter was validated by transfection of minigenes. The six novel mutations expand the mutation spectrum for PFE from eight to 14 pathogenic mutations. Loss-of-function mutations in PTH1R are also associated with recessively inherited Blomstrand chondrodysplasia. We compiled all published PTH1R mutations and identified a mutational overlap between Blomstrand chondrodysplasia and PFE. The results suggest that a genetic approach to preclinical diagnosis will have important implication for surgical and orthodontic treatment of patients with failure of tooth eruption.

  12. Identification of six novel PTH1R mutations in families with a history of primary failure of tooth eruption.

    Directory of Open Access Journals (Sweden)

    Lotte Risom

    Full Text Available Primary Failure of tooth Eruption (PFE is a non-syndromic disorder which can be caused by mutations in the parathyroid hormone receptor 1 gene (PTH1R. Traditionally, the disorder has been identified clinically based on post-emergent failure of eruption of permanent molars. However, patients with PTH1R mutations will not benefit from surgical and/or orthodontic treatment and it is therefore clinically important to establish whether a given failure of tooth eruption is caused by a PTH1R defect or not. We analyzed the PTH1R gene in six patients clinically diagnosed with PFE, all of which had undergone surgical and/or orthodontic interventions, and identified novel PTH1R mutations in all. Four of the six mutations were predicted to abolish correct mRNA maturation either through introduction of premature stop codons (c.947C>A and c.1082G>A, or by altering correct mRNA splicing (c.544-26_544-23del and c.989G>T. The latter was validated by transfection of minigenes. The six novel mutations expand the mutation spectrum for PFE from eight to 14 pathogenic mutations. Loss-of-function mutations in PTH1R are also associated with recessively inherited Blomstrand chondrodysplasia. We compiled all published PTH1R mutations and identified a mutational overlap between Blomstrand chondrodysplasia and PFE. The results suggest that a genetic approach to preclinical diagnosis will have important implication for surgical and orthodontic treatment of patients with failure of tooth eruption.

  13. Identification of dominant flow structures in rapidly rotating convection of liquid metals using Dynamic Mode Decomposition

    Science.gov (United States)

    Horn, S.; Schmid, P. J.; Aurnou, J. M.

    2016-12-01

    The Earth's metal core acts as a dynamo whose efficiency in generating and maintaining the magnetic field is essentially determined by the rotation rate and the convective motions occurring in its outer liquid part. For the description of the primary physics in the outer core the idealized system of rotating Rayleigh-Bénard convection is often invoked, with the majority of studies considering only working fluids with Prandtl numbers of Pr ≳ 1. However, liquid metals are characterized by distinctly smaller Prandtl numbers which in turn result in an inherently different type of convection. Here, we will present results from direct numerical simulations of rapidly rotating convection in a fluid with Pr ≈ 0.025 in cylindrical containers and Ekman numbers as low as 5 × 10-6. In this system, the Coriolis force is the source of two types of inertial modes, the so-called wall modes, that also exist at moderate Prandtl numbers, and cylinder-filling oscillatory modes, that are a unique feature of small Prandtl number convection. The obtained flow fields were analyzed using the Dynamic Mode Decomposition (DMD). This technique allows to extract and identify the structures that govern the dynamics of the system as well as their corresponding frequencies. We have investigated both the regime where the flow is purely oscillatory and the regime where wall modes and oscillatory modes co-exist. In the purely oscillatory regime, high and low frequency oscillatory modes characterize the flow. When both types of modes are present, the DMD reveals that the wall-attached modes dominate the flow dynamics. They precess with a relatively low frequency in retrograde direction. Nonetheless, also in this case, high frequency oscillations have a significant contribution.

  14. Identification of three novel OA1 gene mutations identified in three families misdiagnosed with congenital nystagmus and carrier status determination by real-time quantitative PCR assay

    Directory of Open Access Journals (Sweden)

    Hamel Christian

    2003-01-01

    Full Text Available Abstract Background X-linked ocular albinism type 1 (OA1 is caused by mutations in OA1 gene, which encodes a membrane glycoprotein localised to melanosomes. OA1 mainly affects pigment production in the eye, resulting in optic changes associated with albinism including hypopigmentation of the retina, nystagmus, strabismus, foveal hypoplasia, abnormal crossing of the optic fibers and reduced visual acuity. Affected Caucasian males usually appear to have normal skin and hair pigment. Results We identified three previously undescribed mutations consisting of two intragenic deletions (one encompassing exon 6, the other encompassing exons 7–8, and a point mutation (310delG in exon 2. We report the development of a new method for diagnosis of heterozygous deletions in OA1 gene based on measurement of gene copy number using real-time quantitative PCR from genomic DNA. Conclusion The identification of OA1 mutations in families earlier reported as families with hereditary nystagmus indicate that ocular albinism type 1 is probably underdiagnosed. Our method of real-time quantitative PCR of OA1 exons with DMD exon as external standard performed on the LightCycler™ allows quick and accurate carrier-status assessment for at-risk females.

  15. Evaluation of HIV-1 rapid tests and identification of alternative testing algorithms for use in Uganda.

    Science.gov (United States)

    Kaleebu, Pontiano; Kitandwe, Paul Kato; Lutalo, Tom; Kigozi, Aminah; Watera, Christine; Nanteza, Mary Bridget; Hughes, Peter; Musinguzi, Joshua; Opio, Alex; Downing, Robert; Mbidde, Edward Katongole

    2018-02-27

    The World Health Organization recommends that countries conduct two phase evaluations of HIV rapid tests (RTs) in order to come up with the best algorithms. In this report, we present the first ever such evaluation in Uganda, involving both blood and oral based RTs. The role of weak positive (WP) bands on the accuracy of the individual RT and on the algorithms was also investigated. In total 11 blood based and 3 oral transudate kits were evaluated. All together 2746 participants from seven sites, covering the four different regions of Uganda participated. Two enzyme immunoassays (EIAs) run in parallel were used as the gold standard. The performance and cost of the different algorithms was calculated, with a pre-determined price cut-off of either cheaper or within 20% price of the current algorithm of Determine + Statpak + Unigold. In the second phase, the three best algorithms selected in phase I were used at the point of care for purposes of quality control using finger stick whole blood. We identified three algorithms; Determine + SD Bioline + Statpak; Determine + Statpak + SD Bioline, both with the same sensitivity and specificity of 99.2% and 99.1% respectively and Determine + Statpak + Insti, with sensitivity and specificity of 99.1% and 99% respectively as having performed better and met the cost requirements. There were 15 other algorithms that performed better than the current one but rated more than the 20% price. None of the 3 oral mucosal transudate kits were suitable for inclusion in an algorithm because of their low sensitivities. Band intensity affected the performance of individual RTs but not the final algorithms. We have come up with three algorithms we recommend for public or Government procurement based on accuracy and cost. In case one algorithm is preferred, we recommend to replace Unigold, the current tie breaker with SD Bioline. We further recommend that all the 18 algorithms that have shown better performance than the current one are made

  16. Identification of a novel mutation in a Chinese family with Nance-Horan syndrome by whole exome sequencing*

    Science.gov (United States)

    Hong, Nan; Chen, Yan-hua; Xie, Chen; Xu, Bai-sheng; Huang, Hui; Li, Xin; Yang, Yue-qing; Huang, Ying-ping; Deng, Jian-lian; Qi, Ming; Gu, Yang-shun

    2014-01-01

    Objective: Nance-Horan syndrome (NHS) is a rare X-linked disorder characterized by congenital nuclear cataracts, dental anomalies, and craniofacial dysmorphisms. Mental retardation was present in about 30% of the reported cases. The purpose of this study was to investigate the genetic and clinical features of NHS in a Chinese family. Methods: Whole exome sequencing analysis was performed on DNA from an affected male to scan for candidate mutations on the X-chromosome. Sanger sequencing was used to verify these candidate mutations in the whole family. Clinical and ophthalmological examinations were performed on all members of the family. Results: A combination of exome sequencing and Sanger sequencing revealed a nonsense mutation c.322G>T (E108X) in exon 1 of NHS gene, co-segregating with the disease in the family. The nonsense mutation led to the conversion of glutamic acid to a stop codon (E108X), resulting in truncation of the NHS protein. Multiple sequence alignments showed that codon 108, where the mutation (c.322G>T) occurred, was located within a phylogenetically conserved region. The clinical features in all affected males and female carriers are described in detail. Conclusions: We report a nonsense mutation c.322G>T (E108X) in a Chinese family with NHS. Our findings broaden the spectrum of NHS mutations and provide molecular insight into future NHS clinical genetic diagnosis. PMID:25091991

  17. Identification of a novel mutation in a Chinese family with Nance-Horan syndrome by whole exome sequencing.

    Science.gov (United States)

    Hong, Nan; Chen, Yan-hua; Xie, Chen; Xu, Bai-sheng; Huang, Hui; Li, Xin; Yang, Yue-qing; Huang, Ying-ping; Deng, Jian-lian; Qi, Ming; Gu, Yang-shun

    2014-08-01

    Nance-Horan syndrome (NHS) is a rare X-linked disorder characterized by congenital nuclear cataracts, dental anomalies, and craniofacial dysmorphisms. Mental retardation was present in about 30% of the reported cases. The purpose of this study was to investigate the genetic and clinical features of NHS in a Chinese family. Whole exome sequencing analysis was performed on DNA from an affected male to scan for candidate mutations on the X-chromosome. Sanger sequencing was used to verify these candidate mutations in the whole family. Clinical and ophthalmological examinations were performed on all members of the family. A combination of exome sequencing and Sanger sequencing revealed a nonsense mutation c.322G>T (E108X) in exon 1 of NHS gene, co-segregating with the disease in the family. The nonsense mutation led to the conversion of glutamic acid to a stop codon (E108X), resulting in truncation of the NHS protein. Multiple sequence alignments showed that codon 108, where the mutation (c.322G>T) occurred, was located within a phylogenetically conserved region. The clinical features in all affected males and female carriers are described in detail. We report a nonsense mutation c.322G>T (E108X) in a Chinese family with NHS. Our findings broaden the spectrum of NHS mutations and provide molecular insight into future NHS clinical genetic diagnosis.

  18. Identification and characterization of novel mutations of the major Fanconi anemia gene FANCA in the Japanese population.

    Science.gov (United States)

    Yagasaki, Hiroshi; Hamanoue, Satoshi; Oda, Tsukasa; Nakahata, Tatsutoshi; Asano, Shigetaka; Yamashita, Takayuki

    2004-12-01

    Fanconi anemia (FA) is a rare autosomal recessive disorder of hematopoiesis, with at least 11 complementation groups. FANCA, a gene for group A, accounts for the majority of FA patients. Previous studies of FANCA mutations revealed high allelic heterogeneity, frequent occurrence of large deletions, and interpopulation differences. However, systematic mutational analysis, including gene dosage assay to detect large deletions, has not been documented for Asian populations. A newly developed TaqMan quantitative PCR-based gene dosage assay, combined with sequencing of exons and cDNA fragments, allowed for detection of 48 mutant alleles of FANCA in 27 (77%) of 35 unrelated Japanese FA families with no detectable mutations in FANCC or FANCG. We identified 29 different mutations (21 nucleotide substitutions or small deletions/insertions and eight large deletions), at least 20 of which were novel. The FANCA mutational spectrum of the Japanese was different from that of other ethnic groups so far studied. This is the largest scale of mutation analysis of FANCA in the Japanese population. Characterization of these mutations provided new information regarding the mutagenesis mechanisms and structure-function relationship of FANCA. Specifically, our data suggest that diverse mechanisms including nonhomologous recombination as well as Alu-mediated homologous recombination are involved in the generation of large deletions in FANCA. Copyright 2004 Wiley-Liss, Inc.

  19. Identification of FASTKD2 compound heterozygous mutations as the underlying cause of autosomal recessive MELAS-like syndrome.

    Science.gov (United States)

    Yoo, Da Hye; Choi, Young-Chul; Nam, Da Eun; Choi, Sun Seong; Kim, Ji Won; Choi, Byung-Ok; Chung, Ki Wha

    2017-07-01

    Mitochondrial myopathy, encephalopathy, lactic acidosis, and stroke-like episodes (MELAS) is a condition that affects many parts of the body, particularly the brain and muscles. This study examined a Korean MELAS-like syndrome patient with seizure, stroke-like episode, and optic atrophy. Target sequencing of whole mtDNA and 73 nuclear genes identified compound heterozygous mutations p.R205X and p.L255P in the FASTKD2. Each of his unaffected parents has one of the two mutations, and both mutations were not found in 302 controls. FASTKD2 encodes a FAS-activated serine-threonine (FAST) kinase domain 2 which locates in the mitochondrial inner compartment. A FASTKD2 nonsense mutation was once reported as the cause of a recessive infantile mitochondrial encephalomyopathy. The present case showed relatively mild symptoms with a late onset age, compared to a previous patient with FASTKD2 mutation, implicating an inter-allelic clinical heterogeneity. Because this study is the second report of an autosomal recessive mitochondrial encephalomyopathy patient with a FASTKD2 mutation, it will extend the phenotypic spectrum of the FASTKD2 mutation. Copyright © 2017. Published by Elsevier B.V.

  20. Identification of a novel mutation in the human growth hormone receptor gene (GHR) in a patient with Laron syndrome.

    Science.gov (United States)

    Gennero, Isabelle; Edouard, Thomas; Rashad, Mona; Bieth, Eric; Conte-Aurio, Françoise; Marin, Françoise; Tauber, Maithé; Salles, Jean Pierre; El Kholy, Mohamed

    2007-07-01

    Deletions and mutations in the growth hormone receptor (GHR) gene are the underlying etiology of Laron syndrome (LS) or growth hormone (GH) insensitivity syndrome (GHIS), an autosomal recessive disease. Most patients are distributed in or originate from Mediterranean and Middle-Eastern countries. Sixty mutations have been described so far. We report a novel mutation in the GHR gene in a patient with LS. Genomic DNA sequencing of exon 5 revealed a TT insertion at nucleotide 422 after codon 122. The insertion resulted in a frameshift introducing a premature termination codon that led to a truncated receptor. We present clinical, biochemical and molecular evidence of LS as the result of this homozygous insertion.

  1. Mutation rates at 42 Y chromosomal short tandem repeats in Chinese Han population in Eastern China.

    Science.gov (United States)

    Wu, Weiwei; Ren, Wenyan; Hao, Honglei; Nan, Hailun; He, Xin; Liu, Qiuling; Lu, Dejian

    2018-01-31

    Mutation analysis of 42 Y chromosomal short tandem repeats (Y-STRs) loci was performed using a sample of 1160 father-son pairs from the Chinese Han population in Eastern China. The results showed that the average mutation rate across the 42 Y-STR loci was 0.0041 (95% CI 0.0036-0.0047) per locus per generation. The locus-specific mutation rates varied from 0.000 to 0.0190. No mutation was found at DYS388, DYS437, DYS448, DYS531, and GATA_H4. DYS627, DYS570, DYS576, and DYS449 could be classified as rapidly mutating Y-STRs, with mutation rates higher than 1.0 × 10 -2 . DYS458, DYS630, and DYS518 were moderately mutating Y-STRs, with mutation rates ranging from 8 × 10 -3 to 1 × 10 -2 . Although the characteristics of the Y-STR mutations were consistent with those in previous studies, mutation rate differences between our data and previous published data were found at some rapidly mutating Y-STRs. The single-copy loci located on the short arm of the Y chromosome (Yp) showed relatively higher mutation rates more frequently than the multi-copy loci. These results will not only extend the data for Y-STR mutations but also be important for kinship analysis, paternal lineage identification, and family relationship reconstruction in forensic Y-STR analysis.

  2. Rapid screening and identification of ACE inhibitors in snake venoms using at-line nanofractionation LC-MS.

    Science.gov (United States)

    Mladic, Marija; de Waal, Tessa; Burggraaff, Lindsey; Slagboom, Julien; Somsen, Govert W; Niessen, Wilfried M A; Manjunatha Kini, R; Kool, Jeroen

    2017-10-01

    This study presents an analytical method for the screening of snake venoms for inhibitors of the angiotensin-converting enzyme (ACE) and a strategy for their rapid identification. The method is based on an at-line nanofractionation approach, which combines liquid chromatography (LC), mass spectrometry (MS), and pharmacology in one platform. After initial LC separation of a crude venom, a post-column flow split is introduced enabling parallel MS identification and high-resolution fractionation onto 384-well plates. The plates are subsequently freeze-dried and used in a fluorescence-based ACE activity assay to determine the ability of the nanofractions to inhibit ACE activity. Once the bioactive wells are identified, the parallel MS data reveals the masses corresponding to the activities found. Narrowing down of possible bioactive candidates is provided by comparison of bioactivity profiles after reversed-phase liquid chromatography (RPLC) and after hydrophilic interaction chromatography (HILIC) of a crude venom. Additional nanoLC-MS/MS analysis is performed on the content of the bioactive nanofractions to determine peptide sequences. The method described was optimized, evaluated, and successfully applied for screening of 30 snake venoms for the presence of ACE inhibitors. As a result, two new bioactive peptides were identified: pELWPRPHVPP in Crotalus viridis viridis venom with IC 50  = 1.1 μM and pEWPPWPPRPPIPP in Cerastes cerastes cerastes venom with IC 50  = 3.5 μM. The identified peptides possess a high sequence similarity to other bradykinin-potentiating peptides (BPPs), which are known ACE inhibitors found in snake venoms.

  3. The IRIDICA BAC BSI Assay: Rapid, Sensitive and Culture-Independent Identification of Bacteria and Candida in Blood

    Science.gov (United States)

    Rothman, Richard E.; Peterson, Stephen; Carroll, Karen C.; Zhang, Sean X.; Avornu, Gideon D.; Rounds, Megan A.; Carolan, Heather E.; Toleno, Donna M.; Moore, David; Hall, Thomas A.; Massire, Christian; Richmond, Gregory S.; Gutierrez, Jose R.; Sampath, Rangarajan; Ecker, David J.; Blyn, Lawrence B.

    2016-01-01

    Bloodstream infection (BSI) and sepsis are rising in incidence throughout the developed world. The spread of multi-drug resistant organisms presents increasing challenges to treatment. Surviving BSI is dependent on rapid and accurate identification of causal organisms, and timely application of appropriate antibiotics. Current culture-based methods used to detect and identify agents of BSI are often too slow to impact early therapy and may fail to detect relevant organisms in many positive cases. Existing methods for direct molecular detection of microbial DNA in blood are limited in either sensitivity (likely the result of small sample volumes) or in breadth of coverage, often because the PCR primers and probes used target only a few specific pathogens. There is a clear unmet need for a sensitive molecular assay capable of identifying the diverse bacteria and yeast associated with BSI directly from uncultured whole blood samples. We have developed a method of extracting DNA from larger volumes of whole blood (5 ml per sample), amplifying multiple widely conserved bacterial and fungal genes using a mismatch- and background-tolerant PCR chemistry, and identifying hundreds of diverse organisms from the amplified fragments on the basis of species-specific genetic signatures using electrospray ionization mass spectrometry (PCR/ESI-MS). We describe the analytical characteristics of the IRIDICA BAC BSI Assay and compare its pre-clinical performance to current standard-of-care methods in a collection of prospectively collected blood specimens from patients with symptoms of sepsis. The assay generated matching results in 80% of culture-positive cases (86% when common contaminants were excluded from the analysis), and twice the total number of positive detections. The described method is capable of providing organism identifications directly from uncultured blood in less than 8 hours. Disclaimer: The IRIDICA BAC BSI Assay is not available in the United States. PMID:27384540

  4. Identification of Maturity-Onset Diabetes of the Young Caused by Glucokinase Mutations Detected Using Whole-Exome Sequencing

    Directory of Open Access Journals (Sweden)

    Eun-Hee Cho

    2017-05-01

    Full Text Available Glucokinase maturity-onset diabetes of the young (GCK-MODY represents a distinct subgroup of MODY that does not require hyperglycemia-lowering treatment and has very few diabetes-related complications. Three patients from two families who presented with clinical signs of GCK-MODY were evaluated. Whole-exome sequencing was performed and the effects of the identified mutations were assessed using bioinformatics tools, such as PolyPhen-2, SIFT, and in silico modeling. We identified two mutations: p.Leu30Pro and p.Ser383Leu. In silico analyses predicted that these mutations result in structural conformational changes, protein destabilization, and thermal instability. Our findings may inform future GCK-MODY diagnosis; furthermore, the two mutations detected in two Korean families with GCK-MODY improve our understanding of the genetic basis of the disease.

  5. Identification of a Novel Androgen Receptor Mutation in a Family With Multiple Components Compatible With the Testicular Dysgenesis Syndrome

    DEFF Research Database (Denmark)

    Lottrup, Grete; Jørgensen, Anne; Nielsen, John E.

    2013-01-01

    showed features consistent with insufficient testis development and TDS.Conclusion: The presence of all hallmarks of TDS, including germ cell cancer, in a family with a novel AR mutation causing a partial decrease in AR function is in line with the concept that reduced androgen signaling may contribute......, cryptorchidism, hypospadias, and testicular cancer, caused by a novel AR mutation.Objective: The aim of this study was to describe the phenotype of the affected males, characterize functionally the novel AR mutation, and discuss the significance of partial androgen insufficiency in the pathogenesis of TDS...... analysis of the mutation in a gene-reporter assay showed a 50% reduction in AR-induced transcriptional activity. The affected males had elevated LH and T in accordance with decreased AR signaling. The histology and immunohistochemical profile of the testis tissue from the 2 patients with testicular cancer...

  6. Classifications within molecular subtypes enables identification of BRCA1/BRCA2 mutation carriers by RNA tumor profiling

    DEFF Research Database (Denmark)

    Larsen, Martin J; Kruse, Torben A; Tan, Qihua

    2013-01-01

    Pathogenic germline mutations in BRCA1 or BRCA2 are detected in less than one third of families with a strong history of breast cancer. It is therefore expected that mutations still remain undetected by currently used screening methods. In addition, a growing number of BRCA1/2 sequence variants...... of unclear pathogen significance are found in the families, constituting an increasing clinical challenge. New methods are therefore needed to improve the detection rate and aid the interpretation of the clinically uncertain variants. In this study we analyzed a series of 33 BRCA1, 22 BRCA2, and 128 sporadic...... tumors by RNA profiling to investigate the classification potential of RNA profiles to predict BRCA1/2 mutation status. We found that breast tumors from BRCA1 and BRCA2 mutation carriers display characteristic RNA expression patterns, allowing them to be distinguished from sporadic tumors. The majority...

  7. Rapid identification and antimicrobial susceptibility testing of positive blood cultures using MALDI-TOF MS and a modification of the standardised disc diffusion test: a pilot study.

    LENUS (Irish Health Repository)

    Fitzgerald, C

    2016-04-27

    In an era when clinical microbiology laboratories are under increasing financial pressure, there is a need for inexpensive, yet effective, rapid microbiology tests. The aim of this study was to evaluate a novel modification of standard methodology for the identification and antimicrobial susceptibility testing (AST) of pathogens in positive blood cultures, reducing the turnaround time of laboratory results by 24 h.

  8. Rapid identification and classification of Listeria spp. and serotype assignment of Listeria monocytogenes using fourier transform-infrared spectroscopy and artificial neural network analysis

    Science.gov (United States)

    The use of Fourier Transform-Infrared Spectroscopy (FT-IR) in conjunction with Artificial Neural Network software, NeuroDeveloper™ was examined for the rapid identification and classification of Listeria species and serotyping of Listeria monocytogenes. A spectral library was created for 245 strains...

  9. Development of a triple hyphenated HPLC-radical scavenging detection-DAD-SPE-NMR system for the rapid identification of antioxidants in complex plant extracts

    NARCIS (Netherlands)

    Pukalskas, A.; Beek, van T.A.; Waard, de P.

    2005-01-01

    A rapid method for the simultaneous detection and identification of radical scavenging compounds in plant extracts was developed by combining an HPLC with on-line radical scavenging using DPPH as a model radical and an HPLC¿DAD¿SPE¿NMR system. Using this method a commercial rosemary extract was

  10. Identification and characterization of a novel XK splice site mutation in a patient with McLeod syndrome.

    Science.gov (United States)

    Arnaud, Lionel; Salachas, François; Lucien, Nicole; Maisonobe, Thierry; Le Pennec, Pierre-Yves; Babinet, Jérôme; Cartron, Jean-Pierre

    2009-03-01

    McLeod syndrome is a rare X-linked neuroacanthocytosis syndrome with hematologic, muscular, and neurologic manifestations. McLeod syndrome is caused by mutations in the XK gene whose product is expressed at the red blood cell (RBC) surface but whose function is currently unknown. A variety of XK mutations has been reported but no clear phenotype-genotype correlation has been found, especially for the point mutations affecting splicing sites. A man suspected of neuroacanthocytosis was evaluated by neurologic examination, electromyography, muscle biopsy, muscle computed tomography, and cerebral magnetic resonance imaging. The McLeod RBC phenotype was disclosed by blood smear and immunohematology analyses and then confirmed at the biochemical level by Western blot analysis. The responsible XK mutation was characterized at the mRNA level by reverse transcription-polymerase chain reaction (PCR), identified by genomic DNA sequencing, and verified by allele-specific PCR. A novel XK splice site mutation (IVS1-1G>A) has been identified in a McLeod patient who has developed hematologic, neuromuscular, and neurologic symptoms. This is the first reported example of a XK point mutation affecting the 3' acceptor splice site of Intron 1, and it was demonstrated that this mutation indeed induces aberrant splicing of XK RNA and lack of XK protein at the RBC membrane. The detailed characterization at the molecular biology level of this novel XK splice site mutation associated with the clinical description of the patient contributes to a better understanding of the phenotype-genotype correlation in the McLeod syndrome.

  11. Rapid Detection and Identification of miRNAs by Surface-Enhanced Raman Spectroscopy Using Hollow Au Nanoflowers Substrates

    Directory of Open Access Journals (Sweden)

    Xiaowei Cao

    2017-01-01

    Full Text Available MicroRNAs (miRNAs are recognized as regulators of gene expression during the biological processes of cells as well as biomarkers of many diseases. Development of rapid and sensitive miRNA profiling methods is crucial for evaluating the pattern of miRNA expression related to normal and diseased states. This work presents a novel hollow Au nanoflowers (HAuNFs substrate for rapid detection and identification of miRNAs by surface-enhanced Raman scattering (SERS spectroscopy. We synthesized the HAuNFs by a seed-mediated growth approach. Then, HAuNFs substrates were fabricated by depositing HAuNFs onto the surfaces of (3-aminopropyltriethoxysilane- (APTES- functionalized ITO glass. The result demonstrated that HAuNFs substrates had very good reproducibility, homogeneous SERS activity, and high SERS effect. The substrates enabled us to successfully obtain the SERS spectra of miR-10a-5p, miR-125a-5p, and miR-196a-5p. The difference spectra among the three kinds of miRNAs were studied to better interpret the spectral differences and identify miRNA expression patterns with high accuracy. The principal component analysis (PCA of the SERS spectra was used to distinguish among the three kinds of miRNAs. Considering its time efficiency, being label-free, and its sensitivity, the SERS based on HAuNFs substrates is very promising for miRNA research and plays an important role in early disease detection and prevention.

  12. [Identification of novel compound heterozygous mutations of USH2A gene in a family with Usher syndrome type II].

    Science.gov (United States)

    Jiang, Haiou; Ge, Chuanqin; Wang, Yiwang; Tang, Genyun; Quan, Qingli

    2015-06-01

    To identify potential mutations in a Chinese family with Usher syndrome type II. Genomic DNA was obtained from two affected and four unaffected members of the family and subjected to amplification of the entire coding sequence and splicing sites of USH2A gene. Mutation detection was conducted by direct sequencing of the PCR products. A total of 100 normal unrelated individuals were used as controls. The patients were identified to be a compound heterozygote for two mutations: c.8272G>T (p.E2758X) in exon 42 from his mother and c.12376-12378ACT>TAA(p.T4126X) in exon 63 of the USH2A gene from his father. Both mutations were not found in either of the two unaffected family members or 100 unrelated controls, and had completely co-segregated with the disease phenotype in the family. Neither mutation has been reported in the HGMD database. The novel compound heterozygous mutations c.8272G>T and c.12376-12378ACT>TAA within the USH2A gene may be responsible for the disease. This result may provide new clues for molecular diagnosis of this disease.

  13. Identification of the Rdl mutation in laboratory and field strains of the cat flea, Ctenocephalides felis (Siphonaptera: Pulicidae).

    Science.gov (United States)

    Bass, Chris; Schroeder, Iris; Turberg, Andreas; Field, Linda M; Williamson, Martin S

    2004-12-01

    In many insect species, resistance to cyclodiene insecticides is caused by amino acid substitutions at a single residue (A302) within the M2 transmembrane region of the gamma-aminobutyric acid (GABA) receptor sub-unit termed Rdl (resistance to dieldrin). These mutations (A302S and A302G) have also been shown to confer varying levels of cross-resistance to fipronil, a phenylpyrazole insecticide with a similar mode of action to cyclodienes. To investigate the possible occurrence of these mutations in the cat flea, Ctenocephalides felis (Bouché), a 176-bp fragment of the cat flea Rdl gene, encompassing the mutation site, was PCR amplified and sequenced from nine laboratory flea strains. The A302S mutation was found in eight of the nine strains analysed, although the relative frequency of the mutant allele varied between strains. Only one strain (R6) was found to be homozygous for the S302 allele in all the individuals tested, and this correlated with previous reports of low-level fipronil resistance in this strain. A PCR-based diagnostic assay, capable of screening individual fleas for this mutation, was developed and used to survey a range of fleas collected at random from veterinary clinics in the UK and USA. The A302S mutation was present at a high frequency in these domestic pet populations. 2004 Society of Chemical Industry.

  14. Rapid accumulation of HIV-1 thymidine analogue mutations and phenotypic impact following prolonged viral failure on zidovudine-based first-line ART in sub-Saharan Africa.

    Science.gov (United States)

    Goodall, Ruth L; Dunn, David T; Nkurunziza, Peter; Mugarura, Lincoln; Pattery, Theresa; Munderi, Paula; Kityo, Cissy; Gilks, Charles; Kaleebu, Pontiano; Pillay, Deenan; Gupta, Ravindra K

    2017-05-01

    Lack of viral load monitoring of ART is known to be associated with slower switch from a failing regimen and thereby higher prevalence of MDR HIV-1. Many countries have continued to use thymidine analogue drugs despite recommendations to use tenofovir in combination with a cytosine analogue and NNRTI as first-line ART. The effect of accumulated thymidine analogue mutations (TAMs) on phenotypic resistance over time has been poorly characterized in the African setting. A retrospective analysis of individuals with ongoing viral failure between weeks 48 and 96 in the NORA (Nevirapine OR Abacavir) study was conducted. We analysed 36 genotype pairs from weeks 48 and 96 of first-line ART (14 treated with zidovudine/lamivudine/nevirapine and 22 treated with zidovudine/lamivudine/abacavir). Phenotypic drug resistance was assessed using the Antivirogram assay (v. 2.5.01, Janssen Diagnostics). At 96 weeks, extensive TAMs (≥3 mutations) were present in 50% and 73% of nevirapine- and abacavir-treated patients, respectively. The mean (SE) number of TAMs accumulating between week 48 and week 96 was 1.50 (0.37) in nevirapine-treated participants and 1.82 (0.26) in abacavir-treated participants. Overall, zidovudine susceptibility of viruses was reduced between week 48 [geometric mean fold change (FC) 1.3] and week 96 (3.4, P  =   0.01). There was a small reduction in tenofovir susceptibility (FC 0.7 and 1.0, respectively, P  =   0.18). Ongoing viral failure with zidovudine-containing first-line ART is associated with rapidly increasing drug resistance that could be mitigated with effective viral load monitoring. © The Author 2017. Published by Oxford University Press on behalf of the British Society for Antimicrobial Chemotherapy.

  15. The rapid-onset dystonia parkinsonism mutation D923N of the Na+, K+-ATPase alpha3 isoform disrupts Na+ interaction at the third Na+ site.

    Science.gov (United States)

    Einholm, Anja Pernille; Toustrup-Jensen, Mads S; Holm, Rikke; Andersen, Jens Peter; Vilsen, Bente

    2010-08-20

    Rapid-onset dystonia parkinsonism (RDP), a rare neurological disorder, is caused by mutation of the neuron-specific alpha3-isoform of Na(+), K(+)-ATPase. Here, we present the functional consequences of RDP mutation D923N. Relative to the wild type, the mutant exhibits a remarkable approximately 200-fold reduction of Na(+) affinity for activation of phosphorylation from ATP, reflecting a defective interaction of the E(1) form with intracellular Na(+). This is the largest effect on Na(+) affinity reported so far for any Na(+), K(+)-ATPase mutant. D923N also affects the interaction with extracellular Na(+) normally driving the E(1)P to E(2)P conformational transition backward. However, no impairment of K(+) binding was observed for D923N, leading to the conclusion that Asp(923) is specifically associated with the third Na(+) site that is selective toward Na(+). The crystal structure of the Na(+), K(+)-ATPase in E(2) form shows that Asp(923) is located in the cytoplasmic half of transmembrane helix M8 inside a putative transport channel, which is lined by residues from the transmembrane helices M5, M7, M8, and M10 and capped by the C terminus, recently found involved in recognition of the third Na(+) ion. Structural modeling of the E(1) form of Na(+), K(+)-ATPase based on the Ca(2+)-ATPase crystal structure is consistent with the hypothesis that Asp(923) contributes to a site binding the third Na(+) ion. These results in conjunction with our previous findings with other RDP mutants suggest that a selective defect in the handling of Na(+) may be a general feature of the RDP disorder.

  16. The expanding spectrum of neurological phenotypes in children with ATP1A3 mutations, Alternating Hemiplegia of Childhood, Rapid-onset Dystonia-Parkinsonism, CAPOS and beyond.

    Science.gov (United States)

    Sweney, Matthew T; Newcomb, Tara M; Swoboda, Kathryn J

    2015-01-01

    ATP1A3 mutations have now been recognized in infants and children presenting with a diverse group of neurological phenotypes, including Rapid-onset Dystonia-Parkinsonism (RDP), Alternating Hemiplegia of Childhood (AHC), and most recently, Cerebellar ataxia, Areflexia, Pes cavus, Optic atrophy, and Sensorineural hearing loss (CAPOS) syndrome. Existing literature on ATP1A3-related disorders in the pediatric population were reviewed, with attention to clinical features and associated genotypes among those with RDP, AHC, or CAPOS syndrome phenotypes. While classically defined phenotypes associated with AHC, RDP, and CAPOS syndromes are distinct, common elements among ATP1A3-related neurological disorders include characteristic episodic neurological symptoms and signs that vary in severity, duration, and frequency of occurrence. Affected children typically present in the context of an acute onset of paroxysmal, episodic neurological symptoms ranging from oculomotor abnormalities, hypotonia, paralysis, dystonia, ataxia, seizure-like episodes, or encephalopathy. Neurodevelopmental delays or persistence of dystonia, chorea, or ataxia after resolution of an initial episode are common, providing important clues for diagnosis. The phenotypic spectrum of ATP1A3-related neurological disorders continues to expand beyond the distinct yet overlapping phenotypes in patients with AHC, RDP, and CAPOS syndromes. ATP1A3 mutation analysis is appropriate to consider in the diagnostic algorithm for any child presenting with episodic or fluctuating ataxia, weakness or dystonia whether they manifest persistence of neurological symptoms between episodes. Additional work is needed to better identify and classify affected patients and develop targeted treatment approaches. Copyright © 2015 Elsevier Inc. All rights reserved.

  17. An evaluation of the Oxoid Biochemical Identification System Campy rapid screening test for Campylobacteraceae and Helicobacter spp.

    Science.gov (United States)

    Hoosain, N; Lastovica, A J

    2009-06-01

    To evaluate the Oxoid Biochemical Identification System (OBIS) Campy test (ID0800M) against Campylobacter; Arcobacter; and other micro-organisms, with similar colonial morphology, for the detection of l-alanine aminopeptidase (l-ALA). The KOH and l-ALA (OBIS and Fluka) tests were carried out on every isolate. The procedures were followed as indicated in the OBIS and Fluka kit instructions. A total of 146 strains of 19 species of Campylobacter, seven strains of Arcobacter butzleri, four Arcobacter butzleri-like strains, 42 strains of 10 species of Helicobacter, 96 Gram-negative and 49 Gram-positive clinical isolates were tested. As expected, Campylobacter and Arcobacter strains were negative, while other Gram-negative bacteria were positive for the l-ALA test. An unexpected finding was that Helicobacter strains, although Gram-negative, were all negative for the l-ALA tests suggesting the absence of l-ALA within this genus. This is a novel finding. The absence of l-ALA was confirmed upon comparison with the available full genomic sequences of Helicobacter on the NCBI database. The OBIS Campy (ID0800M) test kit proved to be rapid and accurate for the presumptive characterization of Campylobacter and Arcobacter. A novel finding was that Helicobacter species also did not have the l-ALA enzyme. The OBIS kit will be useful in diagnostic laboratories for the presumptive diagnosis of Campylobacter, Arcobacter and Helicobacter strains.

  18. HR-MAS NMR for rapid identification of illicit substances in tablets and Blotter papers seized by Police Department

    Energy Technology Data Exchange (ETDEWEB)

    Souza, Luciano F.; Vieira, Tarcísio S.; Lião, Luciano M., E-mail: lucianoliao@ufg.br [Universidade Federal de Goiás (UFG), Goiânia, GO (Brazil). Instituto de Química; Alcantara, Glaucia B. [Universidade Federal de Mato Grosso do Sul (UFMS), Campo Grande, MS (Brazil). Instituto de Química

    2016-07-01

    Illicit substances found in blotter papers and tablets seized by police are traditionally identified and characterized from extracts of these materials. However, the procedures involved in extraction stages can result in artifacts and even contamination of the samples to be analyzed. On the other hand, high-resolution magic angle spinning nuclear magnetic resonance (HR-MAS NMR) is a technique that requires no pretreatment steps, enabling direct analysis of the material, including the analysis of new illegal synthetic psychoactive substances. This study presents and discusses applications of the HR-MAS NMR in the analysis of tablets and blotter papers seized. Additional analysis in solution of the extracts of these materials was performed to compare the obtained spectral resolution signals. The results demonstrated that the HR-MAS NMR allowed the rapid identification of 3,4-methylenedioxy-N-methylcathinone (methylone), 4-methylmethcathinone (mephedrone), 2,5-dimethoxy-4-bromoamphetamine (DOB) and 2-(4-bromo-2,5-dimethoxyphenyl)-N-[(2- methoxyphenyl)methyl]ethanamine (25B-NBOMe) in samples of tablets and blotter papers seized in Goiás State, Brazil. (author)

  19. Rapid Detection and Identification of Overdose Drugs in Saliva by Surface-Enhanced Raman Scattering Using Fused Gold Colloids

    Directory of Open Access Journals (Sweden)

    Frank Inscore

    2011-07-01

    Full Text Available The number of drug-related emergency room visits in the United States doubled from 2004 to 2009 to 4.6 million. Consequently there is a critical need to rapidly identify the offending drug(s, so that the appropriate medical care can be administered. In an effort to meet this need we have been investigating the ability of surface-enhanced Raman spectroscopy (SERS to detect and identify numerous drugs in saliva at ng/mL concentrations within 10 minutes. Identification is provided by matching measured spectra to a SERS library comprised of over 150 different drugs, each of which possess a unique spectrum. Trace detection is provided by fused gold colloids trapped within a porous glass matrix that generate SERS. Speed is provided by a syringe-driven sample system that uses a solid-phase extraction capillary combined with a SERS-active capillary in series. Spectral collection is provided by a portable Raman analyzer. Here we describe successful measurement of representative illicit, prescribed, and over-the-counter drugs by SERS, and 50 ng/mL cocaine in saliva as part of a focused study.

  20. HR-MAS NMR for rapid identification of illicit substances in tablets and Blotter papers seized by Police Department

    International Nuclear Information System (INIS)

    Souza, Luciano F.; Vieira, Tarcísio S.; Lião, Luciano M.; Alcantara, Glaucia B.

    2016-01-01

    Illicit substances found in blotter papers and tablets seized by police are traditionally identified and characterized from extracts of these materials. However, the procedures involved in extraction stages can result in artifacts and even contamination of the samples to be analyzed. On the other hand, high-resolution magic angle spinning nuclear magnetic resonance (HR-MAS NMR) is a technique that requires no pretreatment steps, enabling direct analysis of the material, including the analysis of new illegal synthetic psychoactive substances. This study presents and discusses applications of the HR-MAS NMR in the analysis of tablets and blotter papers seized. Additional analysis in solution of the extracts of these materials was performed to compare the obtained spectral resolution signals. The results demonstrated that the HR-MAS NMR allowed the rapid identification of 3,4-methylenedioxy-N-methylcathinone (methylone), 4-methylmethcathinone (mephedrone), 2,5-dimethoxy-4-bromoamphetamine (DOB) and 2-(4-bromo-2,5-dimethoxyphenyl)-N-[(2- methoxyphenyl)methyl]ethanamine (25B-NBOMe) in samples of tablets and blotter papers seized in Goiás State, Brazil. (author)

  1. Standardisation and evaluation of a quantitative multiplex real-time PCR assay for the rapid identification of Streptococcus pneumoniae

    Directory of Open Access Journals (Sweden)

    Feroze Ahmed Ganaie

    2015-01-01

    Full Text Available Rapid diagnosis of Streptococcus pneumoniae can play a significant role in decreasing morbidity and mortality of infection. The accurate diagnosis of pneumococcal disease is hampered by the difficulties in growing the isolates from clinical specimens and also by misidentification. Molecular methods have gained popularity as they offer improvement in the detection of causative pathogens with speed and ease. The present study aims at validating and standardising the use of 4 oligonucleotide primer-probe sets (pneumolysin [ply], autolysin [lytA], pneumococcal surface adhesion A [psaA] and Spn9802 [DNA fragment] in a single-reaction mixture for the detection and discrimination of S. pneumoniae. Here, we validate a quantitative multiplex real-time PCR (qmPCR assay with a panel consisting of 43 S. pneumoniae and 29 non-pneumococcal isolates, 20 culture positive, 26 culture negative and 30 spiked serum samples. A standard curve was obtained using S. pneumoniae ATCC 49619 strain and glyceraldehyde 3-phosphate dehydrogenase (GAPDH gene was used as an endogenous internal control. The experiment showed high sensitivity with lower limit of detection equivalent to 4 genome copies/µl. The efficiency of the reaction was 100% for ply, lytA, Spn9802 and 97% for psaA. The test showed sensitivity and specificity of 100% with culture isolates and serum specimens. This study demonstrates that qmPCR analysis of sera using 4 oligonucleotide primers appears to be an appropriate method for the genotypic identification of S. pneumoniae infection.

  2. PCR-RFLP on β-tubulin gene for rapid identification of the most clinically important species of Aspergillus.

    Science.gov (United States)

    Nasri, Tuba; Hedayati, Mohammad Taghi; Abastabar, Mahdi; Pasqualotto, Alessandro C; Armaki, Mojtaba Taghizadeh; Hoseinnejad, Akbar; Nabili, Mojtaba

    2015-10-01

    Aspergillus species are important agents of life-threatening infections in immunosuppressed patients. Proper speciation in the Aspergilli has been justified based on varied fungal virulence, clinical presentations, and antifungal resistance. Accurate identification of Aspergillus species usually relies on fungal DNA sequencing but this requires expensive equipment that is not available in most clinical laboratories. We developed and validated a discriminative low-cost PCR-based test to discriminate Aspergillus isolates at the species level. The Beta tubulin gene of various reference strains of Aspergillus species was amplified using the universal fungal primers Bt2a and Bt2b. The PCR products were subjected to digestion with a single restriction enzyme AlwI. All Aspergillus isolates were subjected to DNA sequencing for final species characterization. The PCR-RFLP test generated unique patterns for six clinically important Aspergillus species, including Aspergillus flavus, Aspergillus fumigatus, Aspergillus nidulans, Aspergillus terreus, Aspergillus clavatus and Aspergillus nidulans. The one-enzyme PCR-RFLP on Beta tubulin gene designed in this study is a low-cost tool for the reliable and rapid differentiation of the clinically important Aspergillus species. Copyright © 2015 Elsevier B.V. All rights reserved.

  3. Identification of two novel mutations, PSEN1 E280K and PRNP G127S, in a Malaysian family

    Directory of Open Access Journals (Sweden)

    Ch’ng GS

    2015-09-01

    Full Text Available Gaik-Siew Ch’ng,1,* Seong Soo A An,2,* Sun Oh Bae,2 Eva Bagyinszky,2 SangYun Kim3,41Department of Genetics, Kuala Lumpur Hospital, Malaysia; 2Department of Bionano Technology, Gachon University, 3Department of Neurology, Seoul National University College of Medicine, 4Seoul National University Bundang Hospital, South Korea*These authors contributed equally to this workAbstract: Alzheimer’s disease (AD is the most common form of dementia, which can be categorized into two main forms: early onset AD and late onset AD. The genetic background of early onset AD is well understood, and three genes, the APP, PSEN1, and PSEN2 have been identified as causative genes. In the current study, we tested three siblings from Malaysia who were diagnosed with early onset dementia, as well as their available family members. The family history was positive as their deceased father was similarly affected. Patients were tested for mutations in APP, PSEN1, PSEN2, and PRNP. A novel variant, E280K, was discovered in exon 8 of PSEN1 in the three siblings. In silico analyses with SIFT, SNAP, and PolyPhen2 prediction tools and three-dimensional modeling were performed, and the results suggested that the mutation is probably a pathogenic variant. Two additional pathogenic mutations were previously been described for codon 280, E280A, and E280G, which could support the importance of the E280 residue in the PS1 protein contributing to the pathogenic nature of E280K. Additional ten family members were screened for the E280K mutation, and all of them were negative. Six of them presented with a variety of neuropsychiatric symptoms, including learning disabilities, epilepsy, and schizophrenia, while four family members were asymptomatic. A novel PRNP G127S mutation was found in a step-niece of the three siblings harboring the PSEN1 E280K mutation. In silico predictions for PRNP G127S mutation suggested that this might be possibly a damaging variant. Additional studies to

  4. Identification of mutations associated with pyrethroid resistance in the voltage-gated sodium channel of the tomato leaf miner (Tuta absoluta).

    Science.gov (United States)

    Haddi, Khalid; Berger, Madeleine; Bielza, Pablo; Cifuentes, Dina; Field, Linda M; Gorman, Kevin; Rapisarda, Carmelo; Williamson, Martin S; Bass, Chris

    2012-07-01

    The tomato leaf miner, Tuta absoluta (Lepidoptera) is a significant pest of tomatoes that has undergone a rapid expansion in its range during the past six years and is now present across Europe, North Africa and parts of Asia. One of the main means of controlling this pest is through the use of chemical insecticides. In the current study insecticide bioassays were used to determine the susceptibility of five T. absoluta strains established from field collections from Europe and Brazil to pyrethroids. High levels of resistance to λ cyhalothrin and tau fluvalinate were observed in all five strains tested. To investigate whether pyrethroid resistance was mediated by mutation of the para-type sodium channel in T. absoluta the IIS4-IIS6 region of the para gene, which contains many of the mutation sites previously shown to confer knock down (kdr)-type resistance to pyrethroids across a range of different arthropod species, was cloned and sequenced. This revealed that three kdr/super-kdr-type mutations (M918T, T929I and L1014F), were present at high frequencies within all five resistant strains at known resistance 'hot-spots'. This is the first description of these mutations together in any insect population. High-throughput DNA-based diagnostic assays were developed and used to assess the prevalence of these mutations in 27 field strains from 12 countries. Overall mutant allele frequencies were high (L1014F 0.98, M918T 0.35, T929I 0.60) and remarkably no individual was observed that did not carry kdr in combination with either M918T or T929I. The presence of these mutations at high frequency in T. absoluta populations across much of its range suggests pyrethroids are likely to be ineffective for control and supports the idea that the rapid expansion of this species over the last six years may be in part mediated by the resistance of this pest to chemical insecticides. Crown Copyright © 2012. Published by Elsevier Ltd. All rights reserved.

  5. Identification of two novel KIF5A mutations in hereditary spastic paraplegia associated with mild peripheral neuropathy.

    Science.gov (United States)

    López, Eva; Casasnovas, Carlos; Giménez, Javier; Santamaría, Raúl; Terrazas, Jesús M; Volpini, Víctor

    2015-11-15

    Spastic paraplegia type 10 (SPG10) is a rare form of autosomal dominant hereditary spastic paraplegia (AD-HSP) due to mutations in KIF5A, a gene encoding the neuronal kinesin heavy-chain involved in axonal transport. KIF5A mutations have been associated with a wide clinical spectrum, ranging from pure HSP to isolated peripheral nerve involvement or complicated HSP phenotypes. Most KIF5A mutations are clustered in the motor domain of the protein that is necessary for microtubule interaction. Here we describe two Spanish families with an adult onset complicated AD-HSP in which neurological studies revealed a mild sensory neuropathy. Intention tremor was also present in both families. Molecular genetic analysis identified two novel mutations c.773 C>T and c.833 C>T in the KIF5A gene resulting in the P258L and P278L substitutions respectively. Both were located in the highly conserved kinesin motor domain of the protein which has previously been identified as a hot spot for KIF5A mutations. This study adds to the evidence associating the known occurrence of mild peripheral neuropathy in the adult onset SPG10 type of AD-HSP. Copyright © 2015 Elsevier B.V. All rights reserved.

  6. Identification of a de novo mutation of SOX10 in a Chinese patient with Waardenburg syndrome type IV.

    Science.gov (United States)

    Liang, Fenghe; Zhao, Min; Fan, Lynn; Zhang, Hongyan; Shi, Yang; Han, Rui; Qu, Chunyan

    2016-12-01

    Waardenburg syndrome is a rare genetic disorder, characterized by the association of sensorineural hearing loss and pigmentation abnormalities. Four subtypes have been classified. The present study aimed to analyze the clinical feature and investigate the genetic cause for a Chinese case of Waardenburg type IV (WS4). The patient and his family members were subjected to mutation detection in the candidate gene SOX10 by Sanger sequencing. The patient has the clinical features of WS4, including sensorineural hearing loss, bright blue irides, premature graying of the hair and Hirschsprung disease. A novel heterozygous frameshift mutation, c.752_753ins7 (p.Gly252Alafs*31) in the exon 5 of SOX10 was detected in the patient, but not found in the unaffected family members and 100 normal controls. This mutation results in a premature stop codon 31 amino acid downstream. The novel mutation c.752_753ins7 (p.Gly252Alafs*31) arose de novo and was considered as the cause of WS4 in the proband. This study further characterized the molecular complexity of WS4 and provided a clinical case for genotype-phenotype correlation studies of different phenotypes caused by SOX10 mutations. Copyright © 2016 Elsevier Ireland Ltd. All rights reserved.

  7. Identification and functional analysis of a novel mutation in the SOX10 gene associated with Waardenburg syndrome type IV.

    Science.gov (United States)

    Wang, Hong-Han; Chen, Hong-Sheng; Li, Hai-Bo; Zhang, Hua; Mei, Ling-Yun; He, Chu-Feng; Wang, Xing-Wei; Men, Mei-Chao; Jiang, Lu; Liao, Xin-Bin; Wu, Hong; Feng, Yong

    2014-03-15

    Waardenburg syndrome type IV (WS4) is a rare genetic disorder, characterized by auditory-pigmentary abnormalities and Hirschsprung disease. Mutations of the EDNRB gene, EDN3 gene, or SOX10 gene are responsible for WS4. In the present study, we reported a case of a Chinese patient with clinical features of WS4. In addition, the three genes mentioned above were sequenced in order to identify whether mutations are responsible for the case. We revealed a novel nonsense mutation, c.1063C>T (p.Q355*), in the last coding exon of SOX10. The same mutation was not found in three unaffected family members or 100 unrelated controls. Then, the function and mechanism of the mutation were investigated in vitro. We found both wild-type (WT) and mutant SOX10 p.Q355* were detected at the expected size and their expression levels are equivalent. The mutant protein also localized in the nucleus and retained the DNA-binding activity as WT counterpart; however, it lost its transactivation capability on the MITF promoter and acted as a dominant-negative repressor impairing function of the WT SOX10. Copyright © 2014 Elsevier B.V. All rights reserved.

  8. Identification of Deleterious Mutations in Myostatin Gene of Rohu Carp (Labeo rohita Using Modeling and Molecular Dynamic Simulation Approaches

    Directory of Open Access Journals (Sweden)

    Kiran Dashrath Rasal

    2016-01-01

    Full Text Available The myostatin (MSTN is a known negative growth regulator of skeletal muscle. The mutated myostatin showed a double-muscular phenotype having a positive significance for the farmed animals. Consequently, adequate information is not available in the teleosts, including farmed rohu carp, Labeo rohita. In the absence of experimental evidence, computational algorithms were utilized in predicting the impact of point mutation of rohu myostatin, especially its structural and functional relationships. The four mutations were generated at different positions (p.D76A, p.Q204P, p.C312Y, and p.D313A of MSTN protein of rohu. The impacts of each mutant were analyzed using SIFT, I-Mutant 2.0, PANTHER, and PROVEAN, wherein two substitutions (p.D76A and p.Q204P were predicted as deleterious. The comparative structural analysis of each mutant protein with the native was explored using 3D modeling as well as molecular-dynamic simulation techniques. The simulation showed altered dynamic behaviors concerning RMSD and RMSF, for either p.D76A or p.Q204P substitution, when compared with the native counterpart. Interestingly, incorporated two mutations imposed a significant negative impact on protein structure and stability. The present study provided the first-hand information in identifying possible amino acids, where mutations could be incorporated into MSTN gene of rohu carp including other carps for undertaking further in vivo studies.

  9. Identification of four new mutations in the short-chain acyl-CoA dehydrogenase (SCAD) gene in two patients

    DEFF Research Database (Denmark)

    Gregersen, N; Winter, V S; Corydon, M J

    1998-01-01

    We have shown previously that a variant allele of the short-chain acyl-CoA dehydrogenase ( SCAD ) gene, 625G-->A, is present in homozygous form in 7% of control individuals and in 60% of 135 patients with elevated urinary excretion of ethylmalonic acid (EMA). We have now characterized three disease......-causing mutations (confirmed by lack of enzyme activity after expression in COS-7 cells) and a new susceptibility variant in the SCAD gene of two patients with SCAD deficiency, and investigated their frequency in patients with elevated EMA excretion. The first SCAD-deficient patient was a compound heterozygote...... for two mutations, 274G-->T and 529T-->C. These mutations were not present in 98 normal control alleles, but the 529T-->C mutation was found in one allele among 133 patients with elevated EMA excretion. The second patient carried a 1147C-->T mutation and the 625G-->A polymorphism in one allele...

  10. Identification of the first nonsense CDSN mutation with expression of a truncated protein causing peeling skin syndrome type B.

    Science.gov (United States)

    Mallet, A; Kypriotou, M; George, K; Leclerc, E; Rivero, D; Mazereeuw-Hautier, J; Serre, G; Huber, M; Jonca, N; Hohl, D

    2013-12-01

    Peeling skin disease (PSD), a generalized inflammatory form of peeling skin syndrome, is caused by autosomal recessive nonsense mutations in the corneodesmosin gene (CDSN). To investigate a novel mutation in CDSN. A 50-year-old white woman showed widespread peeling with erythema and elevated serum IgE. DNA sequencing, immunohistochemistry, Western blot and real-time polymerase chain reaction analyses of skin biopsies were performed in order to study the genetics and to characterize the molecular profile of the disease. Histology showed hyperkeratosis and acanthosis of the epidermis, and inflammatory infiltrates in the dermis. DNA sequencing revealed a homozygous mutation leading to a premature termination codon in CDSN: p.Gly142*. Protein analyses showed reduced expression of a 16-kDa corneodesmosin mutant in the upper epidermal layers, whereas the full-length protein was absent. These results are interesting regarding the genotype-phenotype correlations in diseases caused by CDSN mutations. The PSD-causing CDSN mutations identified heretofore result in total corneodesmosin loss, suggesting that PSD is due to full corneodesmosin deficiency. Here, we show for the first time that a mutant corneodesmosin can be stably expressed in some patients with PSD, and that this truncated protein is very probably nonfunctional. © 2013 British Association of Dermatologists.

  11. Identification of a novel mutation in the paired domain of PAX3 in an Iranian family with waardenburg syndrome type I.

    Science.gov (United States)

    Sotirova, V N; Rezaie, T M; Khoshsorour, M M; Sarfarazi, M

    2000-03-01

    Waardenburg syndrome Type I (WS1) is an autosomal dominant disorder that has previously been associated with mutations in the PAX3 gene on the 2q35 region. In this study, we used an Iranian WS1 family with seven affected individuals in three generations. The phenotypic characteristics of the family include sensorineural deafness, dystopia canthorum, hypopigmented skin patches of the upper limbs, congenital white forelock, confluent white eyebrows, nonpigmented iris, poliosis, and hypopigmentation of the retina. Herein, we report a previously unidentified single-base substitution in exon II (C-->T at position 218) that results in a change of serine to leucine (S73L) in this family. This change was not observed in 100 chromosomes of healthy unrelated individuals. This mutation is within the PAX3 paired domain region, a structure that is highly conserved and implicated in DNA binding. This is the first identification of a PAX3 mutation for this phenotype in the Iranian population. This also provides additional confirmation for the involvement of this gene in the etiology of WS1.

  12. Identification of a mutation in the CHAT gene of Old Danish Pointing Dogs affected with congenital myasthenic syndrome

    DEFF Research Database (Denmark)

    Proschowsky, Helle Friis; Flagstad, Annette; Cirera, Susanna

    2007-01-01

    The presence of a recessive inherited muscle disease in Old Danish Pointing Dogs has been well known for years. Comparisons of this disease with myasthenic diseases of other dog breeds and humans have pointed toward a defect in the synthesis of the neurotransmitter acetylcholine possibly due...... to decreased activity of the enzyme choline acetyltransferase. We sequenced exons 5-18 of the gene encoding choline acetyltransferase (CHAT) in 2 affected and 2 unaffected dogs and identified a G to A missense mutation in exon 6. The mutation causes a valine to methionine substitution and segregates...... in agreement with the inheritance of the disease. The mutation was not detected in 50 dogs representing 25 other dog breeds. A DNA test has been developed and is now available to the breeders of Old Danish Pointing Dogs....

  13. Identification of a pathogenic FTO mutation by next-generation sequencing in a newborn with growth retardation and developmental delay.

    Science.gov (United States)

    Daoud, Hussein; Zhang, Dong; McMurray, Fiona; Yu, Andrea; Luco, Stephanie M; Vanstone, Jason; Jarinova, Olga; Carson, Nancy; Wickens, James; Shishodia, Shifali; Choi, Hwanho; McDonough, Michael A; Schofield, Christopher J; Harper, Mary-Ellen; Dyment, David A; Armour, Christine M

    2016-03-01

    A homozygous loss-of-function mutation p.(Arg316Gln) in the fat mass and obesity-associated (FTO) gene, which encodes for an iron and 2-oxoglutarate-dependent oxygenase, was previously identified in a large family in which nine affected individuals present with a lethal syndrome characterised by growth retardation and multiple malformations. To date, no other pathogenic mutation in FTO has been identified as a cause of multiple congenital malformations. We investigated a 21-month-old girl who presented distinctive facial features, failure to thrive, global developmental delay, left ventricular cardiac hypertrophy, reduced vision and bilateral hearing loss. We performed targeted next-generation sequencing of 4813 clinically relevant genes in the patient and her parents. We identified a novel FTO homozygous missense mutation (c.956C>T; p.(Ser319Phe)) in the affected individual. This mutation affects a highly conserved residue located in the same functional domain as the previously characterised mutation p.(Arg316Gln). Biochemical studies reveal that p.(Ser319Phe) FTO has reduced 2-oxoglutarate turnover and N-methyl-nucleoside demethylase activity. Our findings are consistent with previous reports that homozygous mutations in FTO can lead to rare growth retardation and developmental delay syndrome, and further support the proposal that FTO plays an important role in early development of human central nervous and cardiovascular systems. Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://www.bmj.com/company/products-services/rights-and-licensing/

  14. Identification of rare heterozygous missense mutations in FANCA in esophageal atresia patients using next-generation sequencing.

    Science.gov (United States)

    Feng, Yu; Chen, Runsen; Da, Min; Qian, Bo; Mo, Xuming

    2018-06-30

    Esophageal atresia and tracheoesophageal fistula (EA/TEF) are relatively common malformations in newborns, but the etiology of EA/TEF remains unknown. Fanconi anemia (FA) complementation group A (FANCA) is a key component of the FA core complex and is essential for the activation of the DNA repair pathway. The middle region (amino acids 674-1208) of FANCA is required for its interaction with FAAP20. We performed targeted sequencing of this binding region of FANCA (exons 23-36) in 40 EA/TEF patients. We also investigated the effect of the p.A958V mutation on the protein-protein interaction between FANCA and FAAP20 using an in vitro binding assay and co-immunoprecipitation. Immunolocalization analysis was performed to investigate the subcellular localization of FANCA, and tissue sections and immunohistochemistry were used to explore the expression of FANCA. We identified four rare missense variants in the FANCA binding region. FANCA mutations were significantly overrepresented in EA/TEF patients compared with 4300 control subjects from the NHLBI-ESP project (Fisher's exact p = 2.17 × 10 -5 , odds ratio = 31.75). p.A958V, a novel de novo mutation in the FANCA gene, was identified in one patient with EA/TEF. We provide further evidence that the p.A958V mutation reduces the binding affinity of FANCA for FAAP20. Interestingly, the p.A958V mutation impaired the nuclear localization of the FANCA protein expressed in HeLa cells. We found that FANCA was more highly expressed in stratified squamous epithelium than in smooth muscle. In conclusion, mutations in the FANCA gene are associated with EA/TEF in humans. Copyright © 2018. Published by Elsevier B.V.

  15. Efficient Identification of Causal Mutations through Sequencing of Bulked F2 from Two Allelic Bloomless Mutants of Sorghum bicolor

    Directory of Open Access Journals (Sweden)

    Yinping Jiao

    2018-01-01

    Full Text Available Sorghum (Sorghum bicolor Moench, L. plant accumulates copious layers of epi-cuticular wax (EW on its aerial surfaces, to a greater extent than most other crops. EW provides a vapor barrier that reduces water loss, and is therefore considered to be a major determinant of sorghum's drought tolerance. However, little is known about the genes responsible for wax accumulation in sorghum. We isolated two allelic mutants, bloomless40-1 (bm40-1 and bm40-2, from a mutant library constructed from ethyl methane sulfonate (EMS treated seeds of an inbred, BTx623. Both mutants were nearly devoid of the EW layer. Each bm mutant was crossed to the un-mutated BTx623 to generated F2 populations that segregated for the bm phenotype. Genomic DNA from 20 bm F2 plants from each population was bulked for whole genome sequencing. A single gene, Sobic.001G228100, encoding a GDSL-like lipase/acylhydrolase, had unique homozygous mutations in each bulked F2 population. Mutant bm40-1 harbored a missense mutation in the gene, whereas bm40-2 had a splice donor site mutation. Our findings thus provide strong evidence that mutation in this GDSL-like lipase gene causes the bm phenotype, and further demonstrate that this approach of sequencing two independent allelic mutant populations is an efficient method for identifying causal mutations. Combined with allelic mutants, MutMap provides powerful method to identify all causal genes for the large collection of bm mutants in sorghum, which will provide insight into how sorghum plants accumulate such abundant EW on their aerial surface. This knowledge may facilitate the development of tools for engineering drought-tolerant crops with reduced water loss.

  16. Identification of point mutations in clinical Staphylococcus aureus strains that produce small-colony variants auxotrophic for menadione.

    Science.gov (United States)

    Dean, Melissa A; Olsen, Randall J; Long, S Wesley; Rosato, Adriana E; Musser, James M

    2014-04-01

    Staphylococcus aureus small-colony variants (SCVs) are implicated in chronic and relapsing infections that are difficult to diagnose and treat. Despite many years of study, the underlying molecular mechanisms and virulence effect of the small-colony phenotype remain incompletely understood. We sequenced the genomes of five S. aureus SCV strains recovered from human patients and discovered previously unidentified nonsynonymous point mutations in three genes encoding proteins in the menadione biosynthesis pathway. Analysis of genetic revertants and complementation with wild-type alleles confirmed that these mutations caused the SCV phenotype and decreased virulence for mice.

  17. Rapid identification of bacteria in positive blood culture broths by matrix-assisted laser desorption ionization-time of flight mass spectrometry.

    Science.gov (United States)

    Stevenson, Lindsay G; Drake, Steven K; Murray, Patrick R

    2010-02-01

    Matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry is a rapid, accurate method for identifying bacteria and fungi recovered on agar culture media. We report herein a method for the direct identification of bacteria in positive blood culture broths by MALDI-TOF mass spectrometry. A total of 212 positive cultures were examined, representing 32 genera and 60 species or groups. The identification of bacterial isolates by MALDI-TOF mass spectrometry was compared with biochemical testing, and discrepancies were resolved by gene sequencing. No identification (spectral score of blood culture broth. Of the bacteria with a spectral score of > or = 1.7, 162 (95.3%) of 170 isolates were correctly identified. All 8 isolates of Streptococcus mitis were misidentified as being Streptococcus pneumoniae isolates. This method provides a rapid, accurate, definitive identification of bacteria within 1 h of detection in positive blood cultures with the caveat that the identification of S. pneumoniae would have to be confirmed by an alternative test.

  18. A simple method for rapid microbial identification from positive monomicrobial blood culture bottles through matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

    Science.gov (United States)

    Lin, Jung-Fu; Ge, Mao-Cheng; Liu, Tsui-Ping; Chang, Shih-Cheng; Lu, Jang-Jih

    2017-06-30

    Rapid identification of microbes in the bloodstream is crucial in managing septicemia because of its high disease severity, and direct identification from positive blood culture bottles through matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) can shorten the turnaround time. Therefore, we developed a simple method for rapid microbiological identification from positive blood cultures by using MALDI-TOF MS. We modified previously developed methods to propose a faster, simpler and more economical method, which includes centrifugation and hemolysis. Specifically, our method comprises two-stage centrifugation with gravitational acceleration (g) at 600g and 3000g, followed by the addition of a lysis buffer and another 3000g centrifugation. In total, 324 monomicrobial bacterial cultures were identified. The success rate of species identification was 81.8%, which is comparable with other complex methods. The identification success rate was the highest for Gram-negative aerobes (85%), followed by Gram-positive aerobes (78.2%) and anaerobes (67%). The proposed method requires less than 10 min, costs less than US$0.2 per usage, and facilitates batch processing. We conclude that this method is feasible for clinical use in microbiology laboratories, and can serve as a reference for treatments or further complementary diagnostic testing. Copyright © 2017. Published by Elsevier B.V.

  19. Rapid Identification of Microorganisms from Positive Blood Culture by MALDI-TOF MS After Short-Term Incubation on Solid Medium.

    Science.gov (United States)

    Curtoni, Antonio; Cipriani, Raffaella; Marra, Elisa Simona; Barbui, Anna Maria; Cavallo, Rossana; Costa, Cristina

    2017-01-01

    Matrix-assisted laser-desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry (MS) is a useful tool for rapid identification of microorganisms. Unfortunately, its direct application to positive blood culture is still lacking standardized procedures. In this study, we evaluated an easy- and rapid-to-perform protocol for MALDI-TOF MS direct identification of microorganisms from positive blood culture after a short-term incubation on solid medium. This protocol was used to evaluate direct identification of microorganisms from 162 positive monomicrobial blood cultures; at different incubation times (3, 5, 24 h), MALDI-TOF MS assay was performed from the growing microorganism patina. Overall, MALDI-TOF MS concordance with conventional methods at species level was 60.5, 80.2, and 93.8% at 3, 5, and 24 h, respectively. Considering only bacteria, the identification performances at species level were 64.1, 85.0, and 94.1% at 3, 5, and 24 h, respectively. This protocol applied to a commercially available MS typing system may represent, a fast and powerful diagnostic tool for pathogen direct identification and for a promptly and pathogen-driven antimicrobial therapy in selected cases.

  20. A Rapid Identification Method for Calamine Using Near-Infrared Spectroscopy Based on Multi-Reference Correlation Coefficient Method and Back Propagation Artificial Neural Network.

    Science.gov (United States)

    Sun, Yangbo; Chen, Long; Huang, Bisheng; Chen, Keli

    2017-07-01

    As a mineral, the traditional Chinese medicine calamine has a similar shape to many other minerals. Investigations of commercially available calamine samples have shown that there are many fake and inferior calamine goods sold on the market. The conventional identification method for calamine is complicated, therefore as a result of the large scale of calamine samples, a rapid identification method is needed. To establish a qualitative model using near-infrared (NIR) spectroscopy for rapid identification of various calamine samples, large quantities of calamine samples including crude products, counterfeits and processed products were collected and correctly identified using the physicochemical and powder X-ray diffraction method. The NIR spectroscopy method was used to analyze these samples by combining the multi-reference correlation coefficient (MRCC) method and the error back propagation artificial neural network algorithm (BP-ANN), so as to realize the qualitative identification of calamine samples. The accuracy rate of the model based on NIR and MRCC methods was 85%; in addition, the model, which took comprehensive multiple factors into consideration, can be used to identify crude calamine products, its counterfeits and processed products. Furthermore, by in-putting the correlation coefficients of multiple references as the spectral feature data of samples into BP-ANN, a BP-ANN model of qualitative identification was established, of which the accuracy rate was increased to 95%. The MRCC method can be used as a NIR-based method in the process of BP-ANN modeling.

  1. Development of a rapid and simplified protocol for direct bacterial identification from positive blood cultures by using matrix assisted laser desorption ionization time-of- flight mass spectrometry.

    Science.gov (United States)

    Jakovljev, Aleksandra; Bergh, Kåre

    2015-11-06

    Bloodstream infections represent serious conditions carrying a high mortality and morbidity rate. Rapid identification of microorganisms and prompt institution of adequate antimicrobial therapy is of utmost importance for a successful outcome. Aiming at the development of a rapid, simplified and efficient protocol, we developed and compared two in-house preparatory methods for the direct identification of bacteria from positive blood culture flasks (BD BACTEC FX system) by using matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI TOF MS). Both methods employed saponin and distilled water for erythrocyte lysis. In method A the cellular pellet was overlaid with formic acid on the MALDI TOF target plate for protein extraction, whereas in method B the pellet was exposed to formic acid followed by acetonitrile prior to placing on the target plate. Best results were obtained by method A. Direct identification was achieved for 81.9 % and 65.8 % (50.3 % and 26.2 % with scores >2.0) of organisms by method A and method B, respectively. Overall concordance with final identification was 100 % to genus and 97.9 % to species level. By applying a lower cut-off score value, the levels of identification obtained by method A and method B increased to 89.3 % and 77.8 % of organisms (81.9 % and 65.8 % identified with scores >1.7), respectively. Using the lowered score criteria, concordance with final results was obtained for 99.3 % of genus and 96.6 % of species identifications. The reliability of results, rapid performance (approximately 25 min) and applicability of in-house method A have contributed to implementation of this robust and cost-effective method in our laboratory.

  2. Osteogenesis imperfecta type I: second-trimester diagnosis and incidental identification of a dominant COL1A1 deletion mutation in the paucisymptomatic father.

    Science.gov (United States)

    Chen, Chih-Ping; Su, Yi-Ning; Chang, Tung-Yao; Chern, Schu-Rern; Chen, Chen-Yu; Su, Jun-Wei; Wang, Wayseen

    2012-06-01

    To present second-trimester ultrasound and molecular diagnosis for osteogenesis imperfecta (OI) type I in a female fetus and incidental identification of a dominant COL1A1 deletion mutation in her paucisymptomatic father. A 30-year-old, primigravid woman was referred for genetic counseling in the second trimester because of bowing of the fetal lower limbs. She and her husband were non-consanguineous, and there was no family history of skeletal dysplasias. Prenatal ultrasound at 22 weeks of gestation revealed short and curved right femur and left tibia, and a short left fibula. The lengths of other long bones were normal. The husband was 158 cm tall, had blue sclerae, a history of habitual subluxation and dislocation of bilateral elbows and left knee, and an episode of left ulna fracture, and was not aware of his being affected with OI type I. The woman underwent amniocentesis. Cytogenetic analysis revealed a karyotype of 46,XX. Molecular analysis of the amniocytes revealed a heterozygous deletion mutation of c.1064_1068delCTGGT in exon 17 of the COL1A1 gene. By genetic testing the husband was found to carry the same mutation. Despite counseling of favorable outcome for OI type I with the parents, the woman elected to terminate the pregnancy. Postnatal skeletal X-ray findings were consistent with OI type I. Prenatal ultrasound diagnosis of mild forms of OI should include molecular analysis of type I collagen genes in both fetus and parents. Molecular genetic analysis of the family may incidentally identify a collagen gene mutation in the paucisymptomatic affected parent. Copyright © 2012. Published by Elsevier B.V.

  3. Rapid detection and identification of Stachybotrys and Chaetomium species using tissue PCR analysis

    DEFF Research Database (Denmark)

    Lewinska, Anna Malgorzata; Peuhkuri, Ruut Hannele; Rode, Carsten

    2016-01-01

    level is essential for health risk assessment and building remediation. This study focuses on molecular identification of two common indoor fungal genera: Stachybotrys and Chaetomium. This study proposes two new DNA barcode candidates for Stachybotrys and Chaetomium: the gene encoding mitogen activated...... protein kinase (hogA) and the intergenic region between histone 3 and histone 4 (h3-h4) as well as it introduces a rapid - 3.5 h - protocol for direct Stachybotrys and Chaetomium species identification, which bypasses culture cultivation, DNA extraction and DNA sequencing....

  4. Identification of a novel EYA1 splice-site mutation in a Danish branchio-oto-renal syndrome family

    DEFF Research Database (Denmark)

    Henriksen, Ann Marie; Tümer, Zeynep; Tommerup, Niels

    2004-01-01

    Branchio-oto-renal (BOR) syndrome is an autosomal dominant disorder characterized by variable clinical manifestations including branchial fistulae, preauricular pits, ear malformations, hearing impairment, and renal anomalies. BOR is caused by mutations in the genes EYA1 and SIX1. A Danish BOR...

  5. Identification of ribonucleotide reductase mutation causing temperature-sensitivity of herpes simplex virus isolates from whitlow by deep sequencing.

    Science.gov (United States)

    Daikoku, Tohru; Oyama, Yukari; Yajima, Misako; Sekizuka, Tsuyoshi; Kuroda, Makoto; Shimada, Yuka; Takehara, Kazuhiko; Miwa, Naoko; Okuda, Tomoko; Sata, Tetsutaro; Shiraki, Kimiyasu

    2015-06-01

    Herpes simplex virus 2 caused a genital ulcer, and a secondary herpetic whitlow appeared during acyclovir therapy. The secondary and recurrent whitlow isolates were acyclovir-resistant and temperature-sensitive in contrast to a genital isolate. We identified the ribonucleotide reductase mutation responsible for temperature-sensitivity by deep-sequencing analysis.

  6. Identification of a disease-causing mutation in a Chinese patient with retinitis pigmentosa by targeted next-generation sequencing

    DEFF Research Database (Denmark)

    Xiao, Jianping; Guo, Xueqin; Wang, Yong

    2017-01-01

    Purpose: To identify disease-causing mutations in a Chinese patient with retinitis pigmentosa (RP). Methods: A detailed clinical examination was performed on the proband. Targeted next-generation sequencing (NGS) combined with bioinformatics analysis was performed on the proband to detect candidate...

  7. Presymptomatic identification of CDH1 germline mutation in a healthy korean individual with family history of gastric cancer.

    Science.gov (United States)

    Choi, Hyun-Jung; Ki, Chang-Seok; Suh, Soon-Pal; Kim, Jong-Won

    2014-09-01

    Gastric cancer (GC) is one of the most common cancers with high morbidity and mortality. Familial GC is seen in 10% of cases, and approximately 3% of familial GC cases arise owing to hereditary diffuse gastric cancer (HDGC). CDH1, which encodes the protein E-cadherin, is the only gene whose mutations are associated with HDGC. Screening for the familial GC-predisposing gene has been neglected in high-risk countries such as Korea, China, and Japan, where all the cases have been attributed to Helicobacter pylori or other carcinogens. Screening for the GC-causing CDH1 mutation may provide valuable information for genetic counseling, testing, and risk-reduction management for the as-yet unaffected family members. An asymptomatic 44-yr-old Korean male visited our genetic clinic for consultation owing to his family history of GC. Eventually, c.1018A>G in CDH1, a known disease-causing mutation, was found. As of the publication time, the individual is alive without the evidence of GC, and is on surveillance. To our knowledge, this is the first Korean case of presymptomatic detection of CDH1 mutation, and it highlights the importance of genetic screening for individuals with a family history of GC, especially in high-risk geographical areas.

  8. Molecular analysis of lipoid proteinosis: identification of a novel nonsense mutation in the ECM1 gene in a Pakistani family

    Directory of Open Access Journals (Sweden)

    Naeem Muhammad

    2011-07-01

    Full Text Available Abstract Lipoid proteinosis is a rare autosomal recessive disease characterized by cutaneous and mucosal lesions and hoarseness appearing in early childhood that is caused by homozygous or compound heterozygous mutations in the ECM1 gene located on chromosome 1q21. The aim of the study was to investigate the molecular genetic defect underlying lipoid proteinosis in a consanguineous Pakistani family. Methods Genotyping of seven members of the family was performed by amplifying microsatellite markers, tightly linked to the ECM1 gene. To screen for mutations in the ECM1 gene, all of its exons and splice junctions were PCR amplified from genomic DNA and analyzed by SSCP and sequenced directly in an ABI 3130 genetic analyzer. Results The results revealed linkage of the LP family to the ECM1 locus. Sequence analysis of the coding exons and splice junctions of the ECM1 gene revealed a novel homozygous mutation (c.616C > T in exon 6, predicted to replace glutamine with stop codon (p.Q206X at amino acid position 206. Conclusions The finding of a novel mutation in Pakistani family extends the body of evidence that supports the importance of ECM1 gene for the development of lipoid proteinosis.

  9. Identification of two novel compound heterozygous mutations of ADGRV1 in a Chinese family with Usher syndrome type IIC.

    Science.gov (United States)

    Zhang, Nian; Wang, Juan; Liu, Shuting; Liu, Mugen; Jiang, Fagang

    2018-06-08

    To describe the clinical and genetic findings in a Chinese family with three sibs diagnosed with Usher syndrome type IIC. Four members received ophthalmic and otologic tests to ascertain the clinical characteristics. According to the clinical phenotype, we focused attention on a total of 658 genes associated with them. We screened the possible pathogenic mutation sites, used Sanger to exclude the false positive and verified whether there were co-segregated among the family members. Typical fundus features found in the proband supported the diagnosis of retinitis pigmentosa (RP). Audiometric test indicated moderate to severe sensorineural hearing impairment while the vestibular function was normal. Whole-exome sequencing identified the presence of two novel compound heterozygous mutations in ADGRV1, a known gene responsible for Usher syndrome type IIC. Mutationc.15008delG/p.Gly5003AlafsTer13 was inherited from the mother while c.18383_18386dupACAG/p.His6130GlnfsTer84 was inherited from the father, and they were co-segregated with the disease phenotype in the family. The mutations found in our study not only broaden the mutation spectrum of ADGRV1, but also provide assistances for future genetic diagnosis and treatment for Usher syndrome patients.

  10. Clinical and molecular characterization of a Brazilian cohort of campomelic dysplasia patients, and identification of seven new SOX9 mutations

    Directory of Open Access Journals (Sweden)

    Eduardo P. Mattos

    2015-03-01

    Full Text Available Campomelic dysplasia (CD is an autosomal, dominantly inherited, skeletal abnormality belonging to the subgroup of bent bone dysplasias. In addition to bowed lower limbs, CD typically includes the following: disproportionate short stature, flat face, micrognathia, cleft palate, bell-shaped thorax, and club feet. Up to three quarters of 46, XY individuals may be sex-reversed. Radiological signs include scapular and pubic hypoplasia, narrow iliac wings, spaced ischia, and bowed femora and tibiae. Lethal CD is usually due to heterozygous mutations in SOX9, a major regulator of chondrocytic development. We present a detailed clinical and molecular characterization of nine Brazilian CD patients. Infants were either stillborn (n = 2 or died shortly after birth and presented similar phenotypes. Sex-reversal was observed in one of three chromosomally male patients. Sequencing of SOX9 revealed new heterozygous mutations in seven individuals. Six patients had mutations that resulted in premature transcriptional termination, while one infant had a single-nucleotide substitution at the conserved splice-site acceptor of intron 1. No clear genotype-phenotype correlations were observed. This study highlights the diversity of SOX9 mutations leading to lethal CD, and expands the group of known genetic alterations associated with this skeletal dysplasia.

  11. Rapid Identification of the Foodborne Pathogen Trichinella spp. by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry.

    Directory of Open Access Journals (Sweden)

    Anne Mayer-Scholl

    Full Text Available Human trichinellosis occurs through consumption of raw or inadequately processed meat or meat products containing larvae of the parasitic nematodes of the genus Trichinella. Currently, nine species and three genotypes are recognized, of which T. spiralis, T. britovi and T. pseudospiralis have the highest public health relevance. To date, the differentiation of the larvae to the species and genotype level is based primarily on molecular methods, which can be relatively time consuming and labor intensive. Due to its rapidness and ease of use a matrix assisted laser desorption / ionization time of flight mass spectrometry (MALDI-TOF MS reference spectra database using Trichinella strains of all known species and genotypes was created. A formicacid/acetonitrile protein extraction was carried out after pooling 10 larvae of each Trichinella species and genotype. Each sample was spotted 9 times using α-cyano 4-hydoxy cinnamic acid matrix and a MicroFlex LT mass spectrometer was used to acquire 3 spectra (m/z 2000 to 20000 Da from each spot resulting in 27 spectra/species or genotype. Following the spectra quality assessment, Biotyper software was used to create a main spectra library (MSP representing nine species and three genotypes of Trichinella. The evaluation of the spectra generated by MALDI-TOF MS revealed a classification which was comparable to the results obtained by molecular methods. Also, each Trichinella species utilized in this study was distinct and distinguishable with a high confidence level. Further, different conservation methods such as freezing and conservation in alcohol and the host species origin of the isolated larvae did not have a significant influence on the generated spectra. Therefore, the described MALDI-TOF MS can successfully be implemented for both genus and species level identification and represents a major step forward in the use of this technique in foodborne parasitology.

  12. Rapid identification of drug-type strains in Cannabis sativa using loop-mediated isothermal amplification assay.

    Science.gov (United States)

    Kitamura, Masashi; Aragane, Masako; Nakamura, Kou; Watanabe, Kazuhito; Sasaki, Yohei

    2017-01-01

    In Cannabis sativa L., tetrahydrocannabinol (THC) is the primary psychoactive compound and exists as the carboxylated form, tetrahydrocannabinolic acid (THCA). C. sativa is divided into two strains based on THCA content-THCA-rich (drug-type) strains and THCA-poor (fiber-type) strains. Both strains are prohibited by law in many countries including Japan, whereas the drug-type strains are regulated in Canada and some European countries. As the two strains cannot be discriminated by morphological analysis, a simple method for identifying the drug-type strains is required for quality control in legal cultivation and forensic investigation. We have developed a novel loop-mediated isothermal amplification (LAMP) assay for identifying the drug-type strains of C. sativa. We designed two selective LAMP primer sets for on-site or laboratory use, which target the drug-type THCA synthase gene. The LAMP assay was accomplished within approximately 40 min. The assay showed high specificity for the drug-type strains and its sensitivity was the same as or higher than that of conventional polymerase chain reaction. We also showed the effectiveness of melting curve analysis that was conducted after the LAMP assay. The melting temperature values of the drug-type strains corresponded to those of the cloned drug-type THCA synthase gene, and were clearly different from those of the cloned fiber-type THCA synthase gene. Moreover, the LAMP assay with simple sample preparation could be accomplished within 1 h from sample treatment to identification without the need for special devices or techniques. Our rapid, sensitive, specific, and simple assay is expected to be applicable to laboratory and on-site detection.

  13. Identification of GLI Mutations in Patients With Hirschsprung Disease That Disrupt Enteric Nervous System Development in Mice.

    Science.gov (United States)

    Liu, Jessica Ai-Jia; Lai, Frank Pui-Ling; Gui, Hong-Sheng; Sham, Mai-Har; Tam, Paul Kwong-Hang; Garcia-Barcelo, Maria-Mercedes; Hui, Chi-Chung; Ngan, Elly Sau-Wai

    2015-12-01

    Hirschsprung disease is characterized by a deficit in enteric neurons, which are derived from neural crest cells (NCCs). Aberrant hedgehog signaling disrupts NCC differentiation and might cause Hirschsprung disease. We performed genetic analyses to determine whether hedgehog signaling is involved in pathogenesis. We performed deep-target sequencing of DNA from 20 patients with Hirschsprung disease (16 men, 4 women), and 20 individuals without (controls), and searched for mutation(s) in GLI1, GLI2, GLI3, SUFU, and SOX10. Biological effects of GLI mutations were tested in luciferase reporter assays using HeLa or neuroblastoma cell lines. Development of the enteric nervous system was studied in Sufu(f/f), Gli3(Δ699), Wnt1-Cre, and Sox10(NGFP) mice using immunohistochemical and whole-mount staining procedures to quantify enteric neurons and glia and analyze axon fasciculation, respectively. NCC migration was studied using time-lapse imaging. We identified 3 mutations in GLI in 5 patients with Hirschsprung disease but no controls; all lead to increased transcription of SOX10 in cell lines. SUFU, GLI, and SOX10 form a regulatory loop that controls the neuronal vs glial lineages and migration of NCCs. Sufu mutants mice had high Gli activity, due to loss of Sufu, disrupting the regulatory loop and migration of enteric NCCs, leading to defective axonal fasciculation, delayed gut colonization, or intestinal hypoganglionosis. The ratio of enteric neurons to glia correlated inversely with Gli activity. We identified mutations that increase GLI activity in patients with Hirschsprung disease. Disruption of the SUFU-GLI-SOX10 regulatory loop disrupts migration of NCCs and development of the enteric nervous system in mice. Copyright © 2015 AGA Institute. Published by Elsevier Inc. All rights reserved.

  14. Conservation patterns of HIV-1 RT connection and RNase H domains: identification of new mutations in NRTI-treated patients.

    Directory of Open Access Journals (Sweden)

    André F A Santos

    Full Text Available BACKGROUND: Although extensive HIV drug resistance information is available for the first 400 amino acids of its reverse transcriptase, the impact of antiretroviral treatment in C-terminal domains of Pol (thumb, connection and RNase H is poorly understood. METHODS AND FINDINGS: We wanted to characterize conserved regions in RT C-terminal domains among HIV-1 group M subtypes and CRF. Additionally, we wished to identify NRTI-related mutations in HIV-1 RT C-terminal domains. We sequenced 118 RNase H domains from clinical viral isolates in Brazil, and analyzed 510 thumb and connection domain and 450 RNase H domain sequences collected from public HIV sequence databases, together with their treatment status and histories. Drug-naïve and NRTI-treated datasets were compared for intra- and inter-group conservation, and differences were determined using Fisher's exact tests. One third of RT C-terminal residues were found to be conserved among group M variants. Three mutations were found exclusively in NRTI-treated isolates. Nine mutations in the connection and 6 mutations in the RNase H were associated with NRTI treatment in subtype B. Some of them lay in or close to amino acid residues which contact nucleic acid or near the RNase H active site. Several of the residues pointed out herein have been recently associated to NRTI exposure or increase drug resistance to NRTI. CONCLUSIONS: This is the first comprehensive genotypic analysis of a large sequence dataset that describes NRTI-related mutations in HIV-1 RT C-terminal domains in vivo. The findings into the conservation of RT C-terminal domains may pave the way to more rational drug design initiatives targeting those regions.

  15. Rapid identification of bacteria from positive blood culture bottles by use of matrix-assisted laser desorption-ionization time of flight mass spectrometry fingerprinting.

    Science.gov (United States)

    Christner, Martin; Rohde, Holger; Wolters, Manuel; Sobottka, Ingo; Wegscheider, Karl; Aepfelbacher, Martin

    2010-05-01

    Early and adequate antimicrobial therapy has been shown to improve the clinical outcome in bloodstream infections (BSI). To provide rapid pathogen identification for targeted treatment, we applied matrix-assisted laser desorption-ionization time of flight (MALDI-TOF) mass spectrometry fingerprinting to bacteria directly recovered from blood culture bottles. A total of 304 aerobic and anaerobic blood cultures, reported positive by a Bactec 9240 system, were subjected in parallel to differential centrifugation with subsequent mass spectrometry fingerprinting and reference identification using established microbiological methods. A representative spectrum of bloodstream pathogens was recovered from 277 samples that grew a single bacterial isolate. Species identification by direct mass spectrometry fingerprinting matched reference identification in 95% of these samples and worked equally well for aerobic and anaerobic culture bottles. Application of commonly used score cutoffs to classify the fingerprinting results led to an identification rate of 87%. Mismatching mostly resulted from insufficient bacterial numbers and preferentially occurred with Gram-positive samples. The respective spectra showed low concordance to database references and were effectively rejected by score thresholds. Spiking experiments and examination of the respective study samples even suggested applicability of the method to mixed cultures. With turnaround times around 100 min, the approach allowed for reliable pathogen identification at the day of blood culture positivity, providing treatment-relevant information within the critical phase of septic illness.

  16. Identification and functional characterisation of novel glucokinase mutations causing maturity-onset diabetes of the young in Slovakia.

    Directory of Open Access Journals (Sweden)

    Lucia Valentínová

    Full Text Available Heterozygous glucokinase (GCK mutations cause a subtype of maturity-onset diabetes of the young (GCK-MODY. Over 600 GCK mutations have been reported of which ∼65% are missense. In many cases co-segregation has not been established and despite the importance of functional studies in ascribing pathogenicity for missense variants these have only been performed for C, c.1113-1114delGC were novel. Parental DNA was available for 22 probands (covering 14/22 mutations and co-segregation established in all cases. Bioinformatic analysis predicted all missense mutations to be damaging. Nine (I110N, V200A, N204D, G223S, G258R, F419S, V244G, L315H, I436N mutations were functionally evaluated. Basic kinetic analysis explained pathogenicity for 7 mutants which showed reduced glucokinase activity with relative activity indices (RAI between 0.6 to <0.001 compared to wild-type GCK (1.0. For the remaining 2 mutants additional molecular mechanisms were investigated. Differences in glucokinase regulatory protein (GKRP -mediated-inhibition of GCK were observed for both L315H & I436N when compared to wild type (IC(50 14.6±0.1 mM & 20.3±1.6 mM vs.13.3±0.1 mM respectively [p<0.03]. Protein instability as assessed by thermal lability studies demonstrated that both L315H and I436N show marked thermal instability compared to wild-type GCK (RAI at 55°C 8.8±0.8% & 3.1±0.4% vs. 42.5±3.9% respectively [p<0.001]. The minimum prevalence of GCK-MODY amongst Slovakian patients with diabetes was 0.03%. In conclusion, we have identified 22 GCK mutations in 36 Slovakian probands and demonstrate that combining family, bioinformatic and functional studies can aid the interpretation of variants identified by molecular diagnostic screening.

  17. Rapid Identification of Dengue Virus by Reverse Transcription-Polymerase Chain Reaction Using Field-Deployable Instrumentation

    National Research Council Canada - National Science Library

    McAvin, James C; Escamilla, Elizabeth M; Blow, James A; Turell, Micahel J; Quintana, Miguel; Bowles, David E; Swaby, James A; Barnes, William J; Huff, William B; Lahman, Kenton L

    2005-01-01

    ...) reverse transcription-polymerase chain reaction assays were developed for screening and seroype identification of infected mosquito vectors and human sera using a field-deployable, fluorometric thermocycler...

  18. Identification and functional analysis of a novel mutation in the PAX3 gene associated with Waardenburg syndrome type I.

    Science.gov (United States)

    Niu, Zhijie; Li, Jiada; Tang, Fen; Sun, Jie; Wang, Xueping; Jiang, Lu; Mei, Lingyun; Chen, Hongsheng; Liu, Yalan; Cai, Xinzhang; Feng, Yong; He, Chufeng

    2018-02-05

    Waardenburg syndrome type 1 (WS1) is a rare autosomal dominant genetic disorder of neural crest cells (NCC) characterized by congenital sensorineural hearing loss, dystopia canthorum, and abnormal iris pigmentation. WS1 is due to loss-of-function mutations in paired box gene 3 (PAX3). Here, we identified a novel PAX3 mutation (c.808C>G, p.R270G) in a three-generation Chinese family with WS1, and then analyzed its in vitro activities. The R270G PAX3 retained nuclear distribution and normal DNA-binding ability; however, it failed to activate MITF promoter, suggesting that haploinsufficiency may be the underlying mechanism for the mild WS1 phenotype of the study family. Copyright © 2017 Elsevier B.V. All rights reserved.

  19. Molecular basis of hereditary C1q deficiency-revisited: identification of several novel disease-causing mutations

    DEFF Research Database (Denmark)

    Schejbel, L; Skattum, L; Hagelberg, S

    2011-01-01

    C1q is the central pattern-recognition molecule in the classical pathway of the complement system and is known to have a key role in the crossroads between adaptive and innate immunity. Hereditary C1q deficiency is a rare genetic condition strongly associated with systemic lupus erythematosus...... and increased susceptibility to bacterial infections. However, the clinical symptoms may vary. For long, the molecular basis of C1q deficiency was ascribed to only six different mutations. In the present report, we describe five new patients with C1q deficiency, present the 12 causative mutations described till...... now and review the clinical spectrum of symptoms found in patients with C1q deficiency. With the results presented here, confirmed C1q deficiency is reported in 64 patients from at least 38 families....

  20. A Rapid and Reproducible Genomic DNA Extraction Protocol for Sequence-Based Identification of Archaea, Bacteria, Cyanobacteria, Diatoms, Fungi, and Green Algae

    OpenAIRE

    Farkhondeh Saba; Moslem Papizadeh; Javad Khansha; Mahshid Sedghi; Mehrnoosh Rasooli; Mohammad Ali Amoozegar; Mohammad Reza Soudi; Seyed Abolhassan Shahzadeh Fazeli

    2016-01-01

    Background:  Sequence-based identification of various microorganisms including Archaea, Bacteria, Cyanobacteria, Diatoms, Fungi, and green algae necessitates an efficient and reproducible genome extraction procedure though which a pure template DNA is yielded and it can be used in polymerase chain reactions (PCR). Considering the fact that DNA extraction from these microorganisms is time consuming and laborious, we developed and standardized a safe, rapid and inexpensive miniprep protocol. Me...

  1. Rapid identification of Campylobacter, Arcobacter, and Helicobacter isolates by PCR-restriction fragment length polymorphism analysis of the 16S rRNA gene.

    Science.gov (United States)

    Marshall, S M; Melito, P L; Woodward, D L; Johnson, W M; Rodgers, F G; Mulvey, M R

    1999-12-01

    A rapid two-step identification scheme based on PCR-restriction fragment length polymorphism (PCR-RFLP) analysis of the 16S rRNA gene was developed in order to differentiate isolates belonging to the Campylobacter, Arcobacter, and Helicobacter genera. For 158 isolates (26 reference cultures and 132 clinical isolates), specific RFLP patterns were obtained and species were successfully identified by this assay.

  2. Identification of a consistent pattern of mutations in neurovirulent variants derived from the sabin vaccine strain of poliovirus type 2.

    OpenAIRE

    Equestre, M; Genovese, D; Cavalieri, F; Fiore, L; Santoro, R; Perez Bercoff, R

    1991-01-01

    Complete nucleotide sequencing of the RNAs of two unrelated neurovirulent isolates of Sabin-related poliovirus type 2 revealed that two nucleotides and one amino acid (amino acid 143 in the major capsid protein VP1) consistently departed from the sequences of the nonneurovirulent poliovirus type 2 712 and Sabin vaccine strains. This pattern of mutation appeared to be a feature common to all neurovirulent variants of poliovirus type 2.

  3. Identification of Novel Voltage-Gated Sodium Channel Mutations in Human Head and Body Lice (Phthiraptera: Pediculidae).

    Science.gov (United States)

    Firooziyan, Samira; Sadaghianifar, Ali; Taghilou, Behrooz; Galavani, Hossein; Ghaffari, Eslam; Gholizadeh, Saber

    2017-09-01

    In recent years, the increase of head louse infestation in Iran (7.4%) and especially in West-Azerbaijan Province (248%) has raised the hypothesis of insecticide resistance development. There are different mechanisms of resistance to various groups of insecticides, and knockdown resistance (kdr) is a prominent mechanism of resistance to pyrethroids, an insecticide group which is used conventionally for pediculosis control. For detection of kdr-type well-known amino acid substitutions (M815I-T917I-L920F) and additional sodium channel mutations potentially associated with kdr resistance in head and body lice, louse populations were collected from West-Azerbaijan and Zanjan Provinces of Iran. Six novel mutations were found to be located in the IIS1-2 extracellular loop (H813P) and IIS5 (I927F, L928A, R929V, L930M, and L932M) of the α-subunit. Genotyping results showed that all specimens (100%) have at least one of these or the well-known mutations. Therefore, the presence of kdr-related and novel mutations in the sodium channel is likely to be the reason for the frequent use of pyrethroid insecticides due to treatment failure against lice. Further studies are now required to evaluate the prevalence of the kdr-like mutant allele for monitoring of insecticide resistance and the management of head and body lice in other provinces of the country. © The Authors 2017. Published by Oxford University Press on behalf of Entomological Society of America. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  4. Identification a novel MYOC gene mutation in a Chinese family with juvenile-onset open angle glaucoma.

    Science.gov (United States)

    Zhao, Xin; Yang, Chaoshan; Tong, Yi; Zhang, Xiaohui; Xu, Liang; Li, Yang

    2010-08-25

    To describe the clinical and genetic findings in one Chinese family with juvenile-onset open angle glaucoma (JOAG). One family was examined clinically and a follow-up took place 5 years later. After informed consent was obtained, genomic DNA was extracted from the venous blood of all participants. Linkage analysis was performed with three microsatellite markers around the MYOC gene (D1S196, D1S2815, and D1S218) in the family. Mutation screening of all coding exons of MYOC was performed by direct sequencing of PCR-amplified DNA fragments and restriction fragment length polymorphism (RFLP) analysis. Bioinformatics analysis by the Garnier-Osguthorpe-Robson (GOR) method predicted the effects of variants detected on secondary structures of the MYOC protein. Clinical examination and pedigree analysis revealed a three- generation family with seven members diagnosed with JOAG, three with ocular hypertension, and five normal individuals. Through genotyping, the pedigree showed a linkage to the MYOC on chromosome 1q24-25. Mutation screening of MYOC in this family revealed an A-->T transition at position 1348 (p. N450Y) of the cDNA sequence. This missense mutation co-segregated with the disease phenotype of the family, but was not found in 100 normal controls. Secondary structure prediction of the p.N450Y by the GOR method revealed the replacement of a coil with a beta sheet at the amino acid 447. Early onset JOAG, with incomplete penetrance, is consistent with a novel mutation in MYOC. The finding provides pre-symptomatic molecular diagnosis for the members of this family and is useful for further genetic consultation.

  5. Identification of new mutations in TCIRG1 as a cause of infantile malignant osteopetrosis in two Mexican patients

    Directory of Open Access Journals (Sweden)

    Claudia Hernández-Martínez

    2018-04-01

    Full Text Available Background: Osteopetrosis is a heterogeneous group of diseases that are characterized by increased bone density due to abnormalities in osteoclast differentiation or function, which result in a lack of bone resorption. Case reports: Two patients with osteopetrosis onset since the first months of life, with facial dysmorphia, blindness, deafness, hepatosplenomegaly, hypotonia, neurodevelopmental retardation and bicytopenia. Bone radiographs showed osteosclerosis. They were assessed by different specialists prior to definitive diagnosis. Genetic analysis determined mutations in the TCIRG1 gene. Patient 1 had a homozygous mutation for p.Ile720Alafs*14 identified, which hasn’t been previously reported. Patient 2 had a compound heterozygous mutation: the first one, p.Phe459Leufs*79, and the second one, p.Gly159Argfs*68, none of which has been previously reported as far as we know. Conclusion: The only therapeutic option for patients with osteopetrosis is hematopoietic stem cell transplantation (HSCT, which should be carried out in the course of the first 3 months of life, before neurological damage occurs. Although osteopetrosis diagnosis is relatively simple, it is delayed owing to the lack of clinical suspicion.

  6. Polyuria and polydipsia in a young child: diagnostic considerations and identification of novel mutation causing familial neurohypophyseal diabetes insipidus.

    Science.gov (United States)

    Stephen, Matthew D; Fenwick, Raymond G; Brosnan, Patrick G

    2012-12-01

    A 3-year 5-month-old boy was seen for second opinion regarding polydipsia and polyuria. Previously, a diagnosis of primary polydipsia was made after normal urine concentration after overnight water deprivation testing. The boy's father, paternal grandfather, and paternal aunt had diabetes insipidus treated with desmopressin acetate. Based on this young boy's symptoms, ability to concentrate urine after informal overnight water deprivation, and family history of diabetes insipidus, we performed AVP gene mutation testing. Analysis of the AVP gene revealed a novel mutation G54E that changes a normal glycine to glutamic acid, caused by a guanine to adenine change at nucleotide g.1537 (exon 2) of the AVP gene. Commonly, patients with familial neurohypophyseal diabetes insipidus (FNHDI) present within the first 6 years of life with progressively worsening polyuria and compensatory polydipsia. Since these patients have progressive loss of arginine vasopressin (AVP), they may initially respond normally to water deprivation testing and have normal pituitary findings on brain MRI. Genetic testing may be helpful in these patients, as well as preemptively diagnosing those with a mutation, thereby avoiding unnecessary surveillance of those unaffected.

  7. Identification of de novo mutations of Duchénnè/Becker muscular dystrophies in southern Spain.

    Science.gov (United States)

    Garcia, Susana; de Haro, Tomás; Zafra-Ceres, Mercedes; Poyatos, Antonio; Gomez-Capilla, Jose A; Gomez-Llorente, Carolina

    2014-01-01

    Duchénnè/Becker muscular dystrophies (DMD/BMD) are X-linked diseases, which are caused by a de novo gene mutation in one-third of affected males. The study objectives were to determine the incidence of DMD/BMD in Andalusia (Spain) and to establish the percentage of affected males in whom a de novo gene mutation was responsible. Multiplex ligation-dependent probe amplification (MLPA) technology was applied to determine the incidence of DMD/BMD in 84 males with suspicion of the disease and 106 female relatives. Dystrophin gene exon deletion (89.5%) or duplication (10.5%) was detected in 38 of the 84 males by MLPA technology; de novo mutations account for 4 (16.7%) of the 24 mother-son pairs studied. MLPA technology is adequate for the molecular diagnosis of DMD/BMD and establishes whether the mother carries the molecular alteration responsible for the disease, a highly relevant issue for genetic counseling.

  8. Rapid identification and typing of Yersinia pestis and other Yersinia species by matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry.

    Science.gov (United States)

    Ayyadurai, Saravanan; Flaudrops, Christophe; Raoult, Didier; Drancourt, Michel

    2010-11-12

    Accurate identification is necessary to discriminate harmless environmental Yersinia species from the food-borne pathogens Yersinia enterocolitica and Yersinia pseudotuberculosis and from the group A bioterrorism plague agent Yersinia pestis. In order to circumvent the limitations of current phenotypic and PCR-based identification methods, we aimed to assess the usefulness of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) protein profiling for accurate and rapid identification of Yersinia species. As a first step, we built a database of 39 different Yersinia strains representing 12 different Yersinia species, including 13 Y. pestis isolates representative of the Antiqua, Medievalis and Orientalis biotypes. The organisms were deposited on the MALDI-TOF plate after appropriate ethanol-based inactivation, and a protein profile was obtained within 6 minutes for each of the Yersinia species. When compared with a 3,025-profile database, every Yersinia species yielded a unique protein profile and was unambiguously identified. In the second step of analysis, environmental and clinical isolates of Y. pestis (n = 2) and Y. enterocolitica (n = 11) were compared to the database and correctly identified. In particular, Y. pestis was unambiguously identified at the species level, and MALDI-TOF was able to successfully differentiate the three biotypes. These data indicate that MALDI-TOF can be used as a rapid and accurate first-line method for the identification of Yersinia isolates.

  9. Development of rapid phenotypic system for the identification of Gram-negative oxidase-positive bacilli in resource-limited settings.

    Science.gov (United States)

    Kazmi, Mahmooda; Khan, Adnan; Kazmi, Shahana Urooj

    2013-06-01

    Rapid and accurate identification of bacterial pathogens is a fundamental goal of clinical microbiology. The diagnosis and surveillance of diseases is dependent, to a great extent, on laboratory services, which cannot function without effective reliable reagents and diagnostics. Despite the advancement in microbiology diagnosis globally, resourcelimited countries still struggle to provide an acceptable diagnosis quality which helps in clinical disease management and improve their mortality and morbidity data. During this study an indigenous product, Quick Test Strip (QTS) NE, was developed for the rapid identification of biochemically slower group of Gram-negative oxidase-positive bacilli that covers 19 different bacterial genera. Some of the members belonging to these groups are well-established human pathogens, e.g. various species of Vibrio, Pseudomonas, Burkholderia, Aeromonas, Achromobacter and Stenotrophomonas. This study also evaluates the performance of QTS-NE by comparing with genotypic characterization methods. A total of 232 clinical and reference bacterial isolates were tested by three different methods. QTSNE provides 100 percent concordant results with other rapid identification and molecular characterization methods and confirms the potential to be used in clinical diagnosis.

  10. Rapid detection and identification of viral and bacterial fish pathogens using a DNA array‐based multiplex assay

    DEFF Research Database (Denmark)

    Lievens, B.; Frans, I.; Heusdens, C.

    2011-01-01

    for the simultaneous detection and identification of all cyprinid herpesviruses (CyHV‐1, CyHV‐2 and CyHV‐3) and some of the most important fish pathogenic Flavobacterium species, including F. branchiophilum, F. columnare and F. psychrophilum. For virus identification, the DNA polymerase and helicase genes were...

  11. Rapidly progressive adenomatous polyposis in a patient with germline mutations in both the APC and MLH1 genes: the worst of two worlds.

    NARCIS (Netherlands)

    Scheenstra, R; Rijcken, FE; Koornstra, JJ; Hollema, H; Fodde, R; Menko, F.H.; Sijmons, RH; Bijleveld, CM; Kleibeuker, J.H.

    2003-01-01

    The two most common inherited forms of colorectal cancer are familial adenomatous polyposis and hereditary non-polyposis colorectal cancer. Simultaneous inheritance of both an APC gene mutation and a mismatch repair gene (for example, MLH1) mutation has never been described. In the present case

  12. [Molecular genetics of pigmentary retinopathies: identification of mutations in CHM, RDS, RHO, RPE65, USH2A and XLRS1 genes].

    Science.gov (United States)

    Hamel, C P; Griffoin, J M; Bazalgette, C; Lasquellec, L; Duval, P A; Bareil, C; Beaufrère, L; Bonnet, S; Eliaou, C; Marlhens, F; Schmitt-Bernard, C F; Tuffery, S; Claustres, M; Arnaud, B

    2000-12-01

    To evaluate the occurrence and inheritance of various types of pigmentary retinopathy in patients followed at the outpatient clinic in the university hospital, Montpellier, France. To characterize genes and mutations causing these conditions. Ophthalmic examination and various visual tests were performed. Mutations were sought from genomic DNA by PCR amplification of exons associated with single-strand conformation analysis and/or direct sequencing. Among 315 patients over an 8-year period, cases of retinitis pigmentosa (63.2%), Usher's syndrome (10.2%), Stargardt's disease (5.4%), choroideremia (3.2%), Leber's congenital amaurosis (3.2%), congenital stationary night blindness (2.9%), cone dystrophy (2.5%), dominant optic atrophy (1.9%), X-linked juvenile retinoschisis (1.6%), Best's disease (1.6%), and others (4.3%) were diagnosed. In retinitis pigmentosa, inheritance could be determined in 54.2% of the cases including dominant autosomic (26.6%), recessive autosomic (22.6%), and X-linked cases (5%) while it could not be confirmed in 45.7% of the cases (simplex cases in the majority). For the 6 examined genes, mutations were found in 22 out of 182 propositus (12.1%). Analysis of phenotype-genotype correlations indicates that in retinitis pigmentosa, RDS is more frequently associated with macular involvement and retinal flecks, RHO with regional disease, and RPE65 with the great severity of the disease with some cases of Leber's congenital amaurosis. Identification of genes may help in diagnosis and in genetic counseling, especially in simplex cases with retinitis pigmentosa. In this latter condition, molecular diagnosis will be necessary to rationalize future treatments.

  13. Rapid identification of bacteria in positive blood culture by matrix-assisted laser desorption ionization time-of-flight mass spectrometry.

    Science.gov (United States)

    Schmidt, V; Jarosch, A; März, P; Sander, C; Vacata, V; Kalka-Moll, W

    2012-03-01

    Blood culture is probably the most significant specimen used for the diagnosis of bacterial infections, especially for bloodstream infections. In the present study, we compared the resin-containing BD BACTEC™ Plus-Aerobic (Becton Dickinson), non-charcoal-containing BacT/Alert(®) SA (bioMérieux), and charcoal-containing BacT/Alert(®) FA (bioMérieux) blood culture bottles with direct identification by matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS). A total of 103 bacterial isolates, from clinical blood cultures, representing the most frequent 13 genera and 24 species were examined. Bacteria were extracted from positive blood culture broth by density centrifugation and then subjected to identification by MALDI-TOF MS using two different volumes and chemical treatments. Overall, correct identification by MALDI-TOF MS was obtained for the BD BACTEC™ Plus-Aerobic, BacT/Alert(®) SA, and BacT/Alert(®) FA blood culture bottles in 72%, 45.6%, and 23%, respectively, for gram-negative bacteria in 86.6%, 69.2%, and 47.1%, respectively, and for gram-positive bacteria in 60.0%, 28.8%, and 5.4%, respectively. The lack of identification was observed mainly with viridans streptococci. Depending on the blood culture bottles used in routine diagnostic procedures and the protocol for bacterial preparation, the applied MALDI-TOF MS represents an efficient and rapid method for direct bacterial identification.

  14. Identification of potential molecular markers of ionizing radiation-induced mutations at the hprt locus in CHO cells

    International Nuclear Information System (INIS)

    Schwartz, J.L.; Sun, J.; Porter, R.C.

    1995-01-01

    Using multiplex polymerase chain reaction-based exon deletion analysis, we have analyzed mutations at the hprt locus from independent CHO cell mutants isolated from untreated, 60 Co x-ray-, and 212 Bi-exposed CHO-K1 cello and its radiation-sensitive derivative, xrs-5. In the 71 spontaneous CHO-K1 mutants analyzed, 78% showed no change in exon number or size, 20% showed loss of 1-8 exons (partial deletion), and 3% showed loss of all nine hprt exons (total deletion). Exposure of CHO-K1 cells to 6 Gy of γ rays (10% survival) produced 45% of the 20 mutants analyzed showing partial deletion, and 30% showing total deletion. Exposure to an equitoxic dose of a radiation from 212 Bi, a 220 Rn daughter, resulted in a spectrum similar to the γ-ray spectrum in that more than 75% of the 49 mutants analyzed were deletions. The α-radiation, however, tended to produce larger intragenic deletions that γ radiation. Of the 87 spontaneous xrs-5 mutants analyzed for deletions 44% showed partial deletion, and 14% showed total deletion. Exposure to α radiation (10% survival) resulted in a deletion spectrum similar to that seen in CHO-K1 cells. Of the 49 mutants analyzed, 43% showed no change in exon number or size, 16% showed partial deletion, and 41% showed total deletion. While the defect in xrs-5 has a profound effect on spontaneous mutation spectra, it does not appear to affect α-induced mutation spectra

  15. Laboratory Evolution of a Biotin-Requiring Saccharomyces cerevisiae Strain for Full Biotin Prototrophy and Identification of Causal Mutations.

    Science.gov (United States)

    Bracher, Jasmine M; de Hulster, Erik; Koster, Charlotte C; van den Broek, Marcel; Daran, Jean-Marc G; van Maris, Antonius J A; Pronk, Jack T

    2017-08-15

    Biotin prototrophy is a rare, incompletely understood, and industrially relevant characteristic of Saccharomyces cerevisiae strains. The genome of the haploid laboratory strain CEN.PK113-7D contains a full complement of biotin biosynthesis genes, but its growth in biotin-free synthetic medium is extremely slow (specific growth rate [μ] ≈ 0.01 h -1 ). Four independent evolution experiments in repeated batch cultures and accelerostats yielded strains whose growth rates (μ ≤ 0.36 h -1 ) in biotin-free and biotin-supplemented media were similar. Whole-genome resequencing of these evolved strains revealed up to 40-fold amplification of BIO1 , which encodes pimeloyl-coenzyme A (CoA) synthetase. The additional copies of BIO1 were found on different chromosomes, and its amplification coincided with substantial chromosomal rearrangements. A key role of this gene amplification was confirmed by overexpression of BIO1 in strain CEN.PK113-7D, which enabled growth in biotin-free medium (μ = 0.15 h -1 ). Mutations in the membrane transporter genes TPO1 and/or PDR12 were found in several of the evolved strains. Deletion of TPO1 and PDR12 in a BIO1 -overexpressing strain increased its specific growth rate to 0.25 h -1 The effects of null mutations in these genes, which have not been previously associated with biotin metabolism, were nonadditive. This study demonstrates that S. cerevisiae strains that carry the basic genetic information for biotin synthesis can be evolved for full biotin prototrophy and identifies new targets for engineering biotin prototrophy into laboratory and industrial strains of this yeast. IMPORTANCE Although biotin (vitamin H) plays essential roles in all organisms, not all organisms can synthesize this vitamin. Many strains of baker's yeast, an important microorganism in industrial biotechnology, contain at least some of the genes required for biotin synthesis. However, most of these strains cannot synthesize biotin at all or do so at rates that are

  16. Laboratory Evolution of a Biotin-Requiring Saccharomyces cerevisiae Strain for Full Biotin Prototrophy and Identification of Causal Mutations

    Science.gov (United States)

    de Hulster, Erik; Koster, Charlotte C.; van den Broek, Marcel; van Maris, Antonius J. A.

    2017-01-01

    ABSTRACT Biotin prototrophy is a rare, incompletely understood, and industrially relevant characteristic of Saccharomyces cerevisiae strains. The genome of the haploid laboratory strain CEN.PK113-7D contains a full complement of biotin biosynthesis genes, but its growth in biotin-free synthetic medium is extremely slow (specific growth rate [μ] ≈ 0.01 h−1). Four independent evolution experiments in repeated batch cultures and accelerostats yielded strains whose growth rates (μ ≤ 0.36 h−1) in biotin-free and biotin-supplemented media were similar. Whole-genome resequencing of these evolved strains revealed up to 40-fold amplification of BIO1, which encodes pimeloyl-coenzyme A (CoA) synthetase. The additional copies of BIO1 were found on different chromosomes, and its amplification coincided with substantial chromosomal rearrangements. A key role of this gene amplification was confirmed by overexpression of BIO1 in strain CEN.PK113-7D, which enabled growth in biotin-free medium (μ = 0.15 h−1). Mutations in the membrane transporter genes TPO1 and/or PDR12 were found in several of the evolved strains. Deletion of TPO1 and PDR12 in a BIO1-overexpressing strain increased its specific growth rate to 0.25 h−1. The effects of null mutations in these genes, which have not been previously associated with biotin metabolism, were nonadditive. This study demonstrates that S. cerevisiae strains that carry the basic genetic information for biotin synthesis can be evolved for full biotin prototrophy and identifies new targets for engineering biotin prototrophy into laboratory and industrial strains of this yeast. IMPORTANCE Although biotin (vitamin H) plays essential roles in all organisms, not all organisms can synthesize this vitamin. Many strains of baker's yeast, an important microorganism in industrial biotechnology, contain at least some of the genes required for biotin synthesis. However, most of these strains cannot synthesize biotin at all or do so at rates

  17. MALDI-TOF mass spectrometry following short incubation on a solid medium is a valuable tool for rapid pathogen identification from positive blood cultures.

    Science.gov (United States)

    Kohlmann, Rebekka; Hoffmann, Alexander; Geis, Gabriele; Gatermann, Sören

    2015-01-01

    Rapid identification of the causative microorganism is a key element in appropriate antimicrobial therapy of bloodstream infections. Whereas traditional analysis of positive blood cultures requires subculture over at least 16-24h prior to pathogen identification by, e.g. matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS), sample preparation procedures enabling direct MALDI-TOF MS, i.e. without preceding subculture, are associated with additional effort and costs. Hence, we integrated an alternative MALDI-TOF MS approach in diagnostic routine using a short incubation on a solid medium. Positive blood cultures were routinely plated on chocolate agar plates and incubated for 4h (37 °C, 5% CO2). Subsequently, MALDI-TOF MS using a Microflex LT instrument (Bruker Daltonics) and direct smear method was performed once per sample. For successful identification of bacteria at species level, score cut-off values were used as proposed by the manufacturer (≥ 2.0) and in a modified form (≥ 1.5 for MALDI-TOF MS results referring to Gram-positive cocci and ≥ 1.7 for MALDI-TOF MS results referring to bacteria other than Gram-positive cocci). Further data analysis also included an assessment of the clinical impact of the MALDI-TOF MS result. Applying the modified score cut-off values, our approach led to an overall correct species identification in 69.5% with misidentification in 3.4% (original cut-offs: 49.2% and 1.8%, respectively); for Gram-positive cocci, correct identification in 68.4% (100% for Staphylococcus aureus and enterococci, 80% for beta-hemolytic streptococci), for Gram-negative bacteria, correct identification in 97.6%. In polymicrobial blood cultures, in 72.7% one of the pathogens was correctly identified. Results were not reliable for Gram-positive rods and yeasts. The approach was easy to implement in diagnostic routine. In cases with available clinical data and successful pathogen identification, in 51.1% our

  18. A novel RT-PCR for the detection of Helicobacter pylori and identification of clarithromycin resistance mediated by mutations in the 23S rRNA gene.

    Science.gov (United States)

    Redondo, Javier Jareño; Keller, Peter M; Zbinden, Reinhard; Wagner, Karoline

    2018-01-01

    In this study we evaluated the commercially available LightMix® RT-PCR assay for Helicobacter pylori detection and identification of clarithromycin (CLR) resistance in culture and clinical specimens (gastric biopsies and stool). The H. pylori LightMix® RT-PCR detects a 97bp long fragment of the 23S rRNA gene and allows the identification of 3 distinct point mutations conferring CLR resistance via melting curve analysis. The performance of the H. pylori LightMix® RT-PCR was evaluated using a set of 60 H. pylori strains showing phenotypical CLR susceptibility or CLR resistance (Minimum inhibitory concentrations from 0.016 to 256mg/L). We found high concordance (95%) between phenotypical CLR resistance screening by E-Test® and the Lightmix® RT-PCR. Discrepant results were verified by sequencing of the 23S rRNA gene that always confirmed the results obtained by Lightmix® RT-PCR. Furthermore, H. pylori was detected in clinical biopsy and stool specimens by Lightmix® RT-PCR that identified the correct H. pylori genotype. The LightMix® RT-PCR is an accurate, sensitive and easy to use test for H. pylori and CLR resistance detection and can therefore be readily implemented in any diagnostic laboratory. Copyright © 2017 The Authors. Published by Elsevier Inc. All rights reserved.

  19. Identification of a missense mutation in the tyrosinase gene in a Chinese family with oculocutaneous albinism type 1.

    Science.gov (United States)

    Lu, Qian; Yuan, Lamei; Xu, Hongbo; Huang, Xiangjun; Yang, Zhijian; Yi, Junhui; Ni, Bin; Chen, Yong; Deng, Hao

    2017-03-01

    Oculocutaneous albinism (OCA) is a group of heterogeneous and autosomal recessive disorders characterized by a reduction or complete loss of melanin biosynthesis in melanocytes. OCA type 1 (OCA1) is the most severe and common form of OCA, and is caused by mutations in the tyrosinase gene (TYR). The present study aimed to identify the genetic cause of OCA1 in a four‑generation consanguineous Chinese Han family. Complete physical examinations were performed and blood samples were collected from five members of the family and 100 unrelated healthy controls. Exome sequencing was conducted in the proband, followed by verification in other family members, using Sanger sequencing. Patients in the family presented with typical OCA1 features, including hypopigmentation of the skin and hair, and distinctive ocular changes. A homozygous missense variant, c.896G>A (p.R299H), in the TYR gene was identified in two patients, which co‑segregated with disease in the family. This variant was not present in the 100 healthy controls. These results expand the number of mutations identified to be responsible for OCA1 in the Chinese Han population, and may have implications for genetic counseling and clinical management of the disease.

  20. Identification of novel resistance mechanisms to NAMPT inhibition via the de novo NAD+ biosynthesis pathway and NAMPT mutation.

    Science.gov (United States)

    Guo, Jun; Lam, Lloyd T; Longenecker, Kenton L; Bui, Mai H; Idler, Kenneth B; Glaser, Keith B; Wilsbacher, Julie L; Tse, Chris; Pappano, William N; Huang, Tzu-Hsuan

    2017-09-23

    Cancer cells have an unusually high requirement for the central and intermediary metabolite nicotinamide adenine dinucleotide (NAD + ), and NAD + depletion ultimately results in cell death. The rate limiting step within the NAD + salvage pathway required for converting nicotinamide to NAD + is catalyzed by nicotinamide phosphoribosyltransferase (NAMPT). Targeting NAMPT has been investigated as an anti-cancer strategy, and several highly selective small molecule inhibitors have been found to potently inhibit NAMPT in cancer cells, resulting in NAD + depletion and cytotoxicity. To identify mechanisms that could cause resistance to NAMPT inhibitor treatment, we generated a human fibrosarcoma cell line refractory to the highly potent and selective NAMPT small molecule inhibitor, GMX1778. We uncovered novel and unexpected mechanisms of resistance including significantly increased expression of quinolinate phosphoribosyl transferase (QPRT), a key enzyme in the de novo NAD + synthesis pathway. Additionally, exome sequencing of the NAMPT gene in the resistant cells identified a single heterozygous point mutation that was not present in the parental cell line. The combination of upregulation of the NAD + de novo synthesis pathway through QPRT over-expression and NAMPT mutation confers resistance to GMX1778, but the cells are only partially resistant to next-generation NAMPT inhibitors. The resistance mechanisms uncovered herein provide a potential avenue to continue exploration of next generation NAMPT inhibitors to treat neoplasms in the clinic. Copyright © 2017 Elsevier Inc. All rights reserved.

  1. Identification of a Novel Mutation in the ABCA4 Gene in a Chinese Family with Retinitis Pigmentosa Using Exome Sequencing.

    Science.gov (United States)

    Huang, Xiangjun; Yuan, Lamei; Xu, Hongbo; Zheng, Wen; Cao, Yanna; Yi, Junhui; Guo, Yi; Yang, Zhijian; Li, Yu; Deng, Hao

    2018-02-05

    Retinitis pigmentosa (RP) is a group of hereditary, degenerative retinal disorders characterized by progressive retinal dysfunction, outer retina cell loss, and retinal tissue atrophy. It eventually leads to tunnel vision and legal, or total blindness. Here we aimed to reveal the causal gene and mutation contributing to the development of autosomal recessive RP (arRP) in a consanguineous family. A novel homozygous mutation, c.4845delT (p.K1616Rfs*46), in the ATP-binding cassette subfamily A member 4gene ( ABCA4 ) was identified. It may reduce ABCA4 protein activity, leading to progressive degeneration of both rod and cone photoreceptors. The study extends the arRP genotypic spectrum and confirms a genotype-phenotype relationship. This study may also disclose some new clues for RP genetic causes and pathogenesis, as well as clinical and genetic diagnosis. The research findings may contribute to improvement in clinical care, therapy, genetic screening, and counseling. ©2018 The Author(s).

  2. The Clinical Impact of Rapid, Direct MALDI-ToF Identification of Bacteria from Positive Blood Cultures.

    Science.gov (United States)

    French, Kathryn; Evans, Jason; Tanner, Hannah; Gossain, Savita; Hussain, Abid

    2016-01-01

    Faster identification of bacterial isolates from blood cultures can enable earlier clinical intervention for patients with sepsis. We evaluated the clinical impact of direct identification of micro-organisms from positive blood cultures using MALDI-ToF. Positive blood cultures with organisms seen on Gram stain were included over a four week period. For each patient case, comparison was made between the clinical advice given on day one with only a Gram stain result, and the follow up advice given on day two with the benefit of organism identification. Culture results were then compared with direct MALDI-ToF identification. For 73 of 115 cases (63.5%), direct organism identification was obtained by MALDI-ToF. Of those 73, 70 (95.5%) had a result concordant with that of the plate culture. In 28 of the 115 cases (24.3%) direct MALDI-ToF identification on day one would have had a clear clinical benefit. In 11 cases it would have helped to identify the potential source of bacteraemia. In 11 cases it would have indicated a different antibiotic regimen on day one, with five patients receiving appropriate antibiotics 24 hours earlier. For 14 cases the blood culture isolate could have been designated as unlikely to be clinically significant. We have demonstrated that organism identification on day one of blood culture positivity can have a direct clinical impact. Faster identification using MALDI-ToF assists the clinician in assessing the significance of a blood culture isolate on day one. It can allow earlier appropriate choice of antimicrobial agent, even in the absence of susceptibility testing, and help narrow down the potential source of infection providing a focus for further investigation in a more timely way than conventional techniques alone.

  3. The Clinical Impact of Rapid, Direct MALDI-ToF Identification of Bacteria from Positive Blood Cultures.

    Directory of Open Access Journals (Sweden)

    Kathryn French

    Full Text Available Faster identification of bacterial isolates from blood cultures can enable earlier clinical intervention for patients with sepsis. We evaluated the clinical impact of direct identification of micro-organisms from positive blood cultures using MALDI-ToF.Positive blood cultures with organisms seen on Gram stain were included over a four week period. For each patient case, comparison was made between the clinical advice given on day one with only a Gram stain result, and the follow up advice given on day two with the benefit of organism identification. Culture results were then compared with direct MALDI-ToF identification.For 73 of 115 cases (63.5%, direct organism identification was obtained by MALDI-ToF. Of those 73, 70 (95.5% had a result concordant with that of the plate culture. In 28 of the 115 cases (24.3% direct MALDI-ToF identification on day one would have had a clear clinical benefit. In 11 cases it would have helped to identify the potential source of bacteraemia. In 11 cases it would have indicated a different antibiotic regimen on day one, with five patients receiving appropriate antibiotics 24 hours earlier. For 14 cases the blood culture isolate could have been designated as unlikely to be clinically significant.We have demonstrated that organism identification on day one of blood culture positivity can have a direct clinical impact. Faster identification using MALDI-ToF assists the clinician in assessing the significance of a blood culture isolate on day one. It can allow earlier appropriate choice of antimicrobial agent, even in the absence of susceptibility testing, and help narrow down the potential source of infection providing a focus for further investigation in a more timely way than conventional techniques alone.

  4. Degenerative myelopathy in German Shepherd Dog: comparison of two molecular assays for the identification of the SOD1:c.118G>A mutation.

    Science.gov (United States)

    Capucchio, Maria Teresa; Spalenza, Veronica; Biasibetti, Elena; Bottero, Maria Teresa; Rasero, Roberto; Dalmasso, Alessandra; Sacchi, Paola

    2014-02-01

    Degenerative myelopathy (DM) is a late-onset, slowly progressive degeneration of spinal cord white matter which is reported primarily in large breed dogs. The missense mutation SOD1:c.118G>A is associated with this pathology in several dog breeds, including the German Shepherd Dog (GSD). The aims of the present study were to develop a tool for the rapid screening of the SOD1 mutation site in dogs and to evaluate the association of the polymorphism with DM in the German Shepherd breed. Two different techniques were compared: a minisequencing test and a real-time pcr allelic discrimination assay. Both approaches resulted effective and efficient. A sample of 47 dogs were examined. Ten subjects presented the symptoms of the illness; for one of them the diagnosis was confirmed by postmortem investigations and it resulted to be an A/A homozygote. In another clinically suspected dog, heterozygote A/G, the histopathological examination of the medulla showed moderate axon and myelin degenerative changes. GSD shows a frequency of the mutant allele equal to 0.17, quite high being a high-risk allele. Because canine DM has a late onset in adulthood and homozygous mutant dogs are likely as fertile as other genotypes, the natural selection is mild and the mutant allele may reach high frequencies. A diagnostic test, easy to implement, may contribute to control the gene diffusion in populations. The SOD1:c.118G>A mutation could be a useful marker for breeding strategies intending to reduce the incidence of DM.

  5. Rapid label-free identification of Klebsiella pneumoniae antibiotic resistant strains by the drop-coating deposition surface-enhanced Raman scattering method

    Science.gov (United States)

    Cheong, Youjin; Kim, Young Jin; Kang, Heeyoon; Choi, Samjin; Lee, Hee Joo

    2017-08-01

    Although many methodologies have been developed to identify unknown bacteria, bacterial identification in clinical microbiology remains a complex and time-consuming procedure. To address this problem, we developed a label-free method for rapidly identifying clinically relevant multilocus sequencing typing-verified quinolone-resistant Klebsiella pneumoniae strains. We also applied the method to identify three strains from colony samples, ATCC70063 (control), ST11 and ST15; these are the prevalent quinolone-resistant K. pneumoniae strains in East Asia. The colonies were identified using a drop-coating deposition surface-enhanced Raman scattering (DCD-SERS) procedure coupled with a multivariate statistical method. Our workflow exhibited an enhancement factor of 11.3 × 106 to Raman intensities, high reproducibility (relative standard deviation of 7.4%), and a sensitive limit of detection (100 pM rhodamine 6G), with a correlation coefficient of 0.98. All quinolone-resistant K. pneumoniae strains showed similar spectral Raman shifts (high correlations) regardless of bacterial type, as well as different Raman vibrational modes compared to Escherichia coli strains. Our proposed DCD-SERS procedure coupled with the multivariate statistics-based identification method achieved excellent performance in discriminating similar microbes from one another and also in subtyping of K. pneumoniae strains. Therefore, our label-free DCD-SERS procedure coupled with the computational decision supporting method is a potentially useful method for the rapid identification of clinically relevant K. pneumoniae strains.

  6. DNA-based identification of invasive alien species in relation to Canadian federal policy and law, and the basis of rapid-response management.

    Science.gov (United States)

    Thomas, Vernon G; Hanner, Robert H; Borisenko, Alex V

    2016-11-01

    Managing invasive alien species in Canada requires reliable taxonomic identification as the basis of rapid-response management. This can be challenging, especially when organisms are small and lack morphological diagnostic features. DNA-based techniques, such as DNA barcoding, offer a reliable, rapid, and inexpensive toolkit for taxonomic identification of individual or bulk samples, forensic remains, and even environmental DNA. Well suited for this requirement, they could be more broadly deployed and incorporated into the operating policy and practices of Canadian federal departments and should be authorized under these agencies' articles of law. These include Fisheries and Oceans Canada, Canadian Food Inspection Agency, Transport Canada, Environment Canada, Parks Canada, and Health Canada. These efforts should be harmonized with the appropriate provisions of provincial jurisdictions, for example, the Ontario Invasive Species Act. This approach necessitates that a network of accredited, certified laboratories exists, and that updated DNA reference libraries are readily accessible. Harmonizing this approach is vital among Canadian federal agencies, and between the federal and provincial levels of government. Canadian policy and law must also be harmonized with that of the USA when detecting, and responding to, invasive species in contiguous lands and waters. Creating capacity in legislation for use of DNA-based identifications brings the authority to fund, train, deploy, and certify staff, and to refine further developments in this molecular technology.

  7. Usher Syndrome Type III: Revised Genomic Structure of the USH3 Gene and Identification of Novel Mutations

    Science.gov (United States)

    Fields, Randall R.; Zhou, Guimei; Huang, Dali; Davis, Jack R.; Möller, Claes; Jacobson, Samuel G.; Kimberling, William J.; Sumegi, Janos

    2002-01-01

    Usher syndrome type III is an autosomal recessive disorder characterized by progressive sensorineural hearing loss, vestibular dysfunction, and retinitis pigmentosa. The disease gene was localized to 3q25 and recently was identified by positional cloning. In the present study, we have revised the structure of the USH3 gene, including a new translation start site, 5′ untranslated region, and a transcript encoding a 232–amino acid protein. The mature form of the protein is predicted to contain three transmembrane domains and 204 residues. We have found four new disease-causing mutations, including one that appears to be relatively common in the Ashkenazi Jewish population. We have also identified mouse (chromosome 3) and rat (chromosome 2) orthologues, as well as two human paralogues on chromosomes 4 and 10. PMID:12145752

  8. Identification of a novel ZIC3 isoform and mutation screening in patients with heterotaxy and congenital heart disease.

    Directory of Open Access Journals (Sweden)

    James E J Bedard

    Full Text Available Patients with heterotaxy have characteristic cardiovascular malformations, abnormal arrangement of their visceral organs, and midline patterning defects that result from abnormal left-right patterning during embryogenesis. Loss of function of the transcription factor ZIC3 causes X-linked heterotaxy and isolated congenital heart malformations and represents one of the few known monogenic causes of congenital heart disease. The birth incidence of heterotaxy-spectrum malformations is significantly higher in males, but our previous work indicated that mutations within ZIC3 did not account for the male over-representation. Therefore, cross species comparative sequence alignment was used to identify a putative novel fourth exon, and the existence of a novel alternatively spliced transcript was confirmed by amplification from murine embryonic RNA and subsequent sequencing. This transcript, termed Zic3-B, encompasses exons 1, 2, and 4 whereas Zic3-A encompasses exons 1, 2, and 3. The resulting protein isoforms are 466 and 456 amino acid residues respectively, sharing the first 407 residues. Importantly, the last two amino acids in the fifth zinc finger DNA binding domain are altered in the Zic3-B isoform, indicating a potential functional difference that was further evaluated by expression, subcellular localization, and transactivation analyses. The temporo-spatial expression pattern of Zic3-B overlaps with Zic3-A in vivo, and both isoforms are localized to the nucleus in vitro. Both isoforms can transcriptionally activate a Gli binding site reporter, but only ZIC3-A synergistically activates upon co-transfection with Gli3, suggesting that the isoforms are functionally distinct. Screening 109 familial and sporadic male heterotaxy cases did not identify pathogenic mutations in the newly identified fourth exon and larger studies are necessary to establish the importance of the novel isoform in human disease.

  9. Brain Malformations Associated with Knobloch Syndrome – Review of Literature, Expanding Clinical Spectrum and Identification of Novel Mutations

    Science.gov (United States)

    Caglayan, Ahmet Okay; Baranoski, Jacob F.; Aktar, Fesih; Han, Wengi; Tuysuz, Beyhan; Guzel, Aslan; Guclu, Bulent; Kaymakcalan, Hande; Aktekin, Berrin; Akgumus, Gozde Tugce; Murray, Phillip B.; Omay, E. Zeynep Erson; Caglar, Caner; Bakircioglu, Mehmet; Sakalar, Yildirim Bayezit; Guzel, Ebru; Demir, Nihat; Tuncer, Oguz; Senturk, Senem; Ekici, Baris; Minja, Frank J.; Šestan, Nenad; Yasuno, Katsuhito; Bilguvar, Kaya; Caksen, Huseyin; Gunel, Murat

    2014-01-01

    BACKGROUND Knobloch syndrome is a rare, autosomal recessive, developmental disorder characterized by stereotyped ocular abnormalities with or without occipital skull deformities (encephalocele, bone defects, cutis aplasia). Although there is clear heterogeneity in clinical presentation, central nervous system malformations, aside from the characteristic encephalocele, have not typically been considered a component of the disease phenotype. METHODS Four patients originally presented for genetic evaluation of symptomatic structural brain malformations. Whole-genome genotyping, whole-exome sequencing, and confirmatory Sanger sequencing were performed. Using immunohistochemical analysis, we investigated the protein expression pattern of COL18A1 in the mid-fetal and adult human cerebral cortex and then analyzed the spatial and temporal changes in the expression pattern of COL18A1 during human cortical development using the Human Brain Transcriptome database. RESULTS We identified two novel homozygous deleterious frame-shift mutations in the COL18A1 gene. Upon further investigation of these patients and their families, we found that many exhibited certain characteristics of Knobloch syndrome, including pronounced ocular defects. Our data strongly support an important role for COL18A1 in brain development and this report contributes to an enhanced characterization of the brain malformations that can result from deficiencies of collagen XVIII. CONCLUSIONS This case series highlights the diagnostic power and clinical utility of whole-exome sequencing technology – allowing clinicians and physician scientists to better understand the pathophysiology and presentations of rare diseases. We suggest that patients who are clinically diagnosed with Knobloch syndrome and/or found to have COL18A1 mutations via genetic screening should be investigated for potential structural brain abnormalities even in the absence of encephaloceles. PMID:25456301

  10. MALDI-TOF MS enables the rapid identification of the major molecular types within the Cryptococcus neoformans/C. gattii species complex.

    Directory of Open Access Journals (Sweden)

    Carolina Firacative

    Full Text Available BACKGROUND: The Cryptococcus neoformans/C. gattii species complex comprises two sibling species that are divided into eight major molecular types, C. neoformans VNI to VNIV and C. gattii VGI to VGIV. These genotypes differ in host range, epidemiology, virulence, antifungal susceptibility and geographic distribution. The currently used phenotypic and molecular identification methods for the species/molecular types are time consuming and expensive. As Matrix-Assisted Laser Desorption Ionization-Time-of-Flight Mass Spectrometry (MALDI-TOF MS offers an effective alternative for the rapid identification of microorganisms, the objective of this study was to examine its potential for the identification of C. neoformans and C. gattii strains at the intra- and inter-species level. METHODOLOGY: Protein extracts obtained via the formic acid extraction method of 164 C. neoformans/C. gattii isolates, including four inter-species hybrids, were studied. RESULTS: The obtained mass spectra correctly identified 100% of all studied isolates, grouped each isolate according to the currently recognized species, C. neoformans and C. gattii, and detected potential hybrids. In addition, all isolates were clearly separated according to their major molecular type, generating greater spectral differences among the C. neoformans molecular types than the C. gattii molecular types, most likely reflecting a closer phylogenetic relationship between the latter. The number of colonies used and the incubation length did not affect the results. No spectra were obtained from intact yeast cells. An extended validated spectral library containing spectra of all eight major molecular types was established. CONCLUSIONS: MALDI-TOF MS is a rapid identification tool for the correct recognition of the two currently recognized human pathogenic Cryptococcus species and offers a simple method for the separation of the eight major molecular types and the detection of hybrid strains within this

  11. Novel, Improved Sample Preparation for Rapid, Direct Identification from Positive Blood Cultures Using Matrix-Assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF) Mass Spectrometry

    OpenAIRE

    Schubert, Sören; Weinert, Kirsten; Wagner, Chris; Gunzl, Beatrix; Wieser, Andreas; Maier, Thomas; Kostrzewa, Markus

    2011-01-01

    Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) is widely used for rapid and reliable identification of bacteria and yeast grown on agar plates. Moreover, MALDI-TOF MS also holds promise for bacterial identification from blood culture (BC) broths in hospital laboratories. The most important technical step for the identification of bacteria from positive BCs by MALDI-TOF MS is sample preparation to remove blood cells and host proteins. We present a m...

  12. Use of positive ion fast atom bombardment mass spectrometry for rapid identification of a bile alcohol glucuronide isolated from cerebrotendinous xanthomatosis patients

    International Nuclear Information System (INIS)

    Dayal, B.; Salen, G.; Tint, G.S.; Shefer, S.; Benz, S.W.

    1990-01-01

    The identification of a major biliary and plasma bile alcohol glucuronide, 5 beta-cholestane-3 alpha, 7 alpha, 12 alpha, 25-tetrol-3-0-beta-D-glucuronide, present in cerebrotendinous xanthomatosis (CTX) patients, was investigated by positive ion fast atom bombardment mass spectrometry (FAB-MS). The spectrum was characterized by abundant ions formed by attachment of a proton, [M + H]+, or of alkali ions, [M + Na]+ and [M + 39K]+, to the glucuronide salt. These ions allowed an unambiguous deduction of the molecular weight of the sample. It is suggested that FAB-MS could be used in the rapid diagnosis of CTX

  13. DNA microarray-based solid-phase RT-PCR for rapid detection and identification of influenza virus type A and subtypes H5 and H7

    DEFF Research Database (Denmark)

    Yi, Sun; Dhumpa, Raghuram; Bang, Dang Duong

    2011-01-01

    of RNA extract in the liquid phase with sequence-specific nested PCR on the solid phase. A simple ultraviolet cross-linking method was used to immobilize the DNA probes over an unmodified glass surface, which makes solid-phase PCR a convenient possibility for AIV screening. The testing of 33 avian fecal....... In this article, a DNA microarray-based solid-phase polymerase chain reaction (PCR) approach has been developed for rapid detection of influenza virus type A and for simultaneous identification of pathogenic virus subtypes H5 and H7. This solid-phase RT-PCR method combined reverse-transcription amplification...

  14. Rapid identification of microorganisms from positive blood cultures by MALDI-TOF mass spectrometry subsequent to very short-term incubation on solid medium.

    Science.gov (United States)

    Idelevich, E A; Schüle, I; Grünastel, B; Wüllenweber, J; Peters, G; Becker, K

    2014-10-01

    Rapid identification of the causative microorganism is important for appropriate antimicrobial therapy of bloodstream infections. Bacteria from positive blood culture (BC) bottles are not readily available for identification by matrix-assisted laser desorption ionization-time of flight mass spectrometry (MALDI-TOF MS). Lysis and centrifugation procedures suggested for direct MALDI-TOF MS from positive BCs without previous culture are associated with additional hands-on processing time and costs. Here, we describe an alternative approach applying MALDI-TOF MS from bacterial cultures incubated very briefly on solid medium. After plating of positive BC broth on Columbia blood agar (n = 165), MALDI-TOF MS was performed after 1.5, 2, 3, 4, 5, 6, 7, 8, 12 and (for control) 24 h of incubation until reliable identification to the species level was achieved (score ≥2.0). Mean incubation time needed to achieve species-level identification was 5.9 and 2.0 h for Gram-positive aerobic cocci (GPC, n = 86) and Gram-negative aerobic rods (GNR, n = 42), respectively. Short agar cultures with incubation times ≤2, ≤4, ≤6, ≤8 and ≤12 h yielded species identification in 1.2%, 18.6%, 64.0%, 96.5%, 98.8% of GPC, and in 76.2%, 95.2%, 97.6%, 97.6%, 97.6% of GNR, respectively. Control species identification at 24 h was achieved in 100% of GPC and 97.6% of GNR. Ethanol/formic acid protein extraction performed for an additional 34 GPC isolates cultivated from positive BCs showed further reduction in time to species identification (3.1 h). MALDI-TOF MS using biomass subsequent to very short-term incubation on solid medium allows very early and reliable bacterial identification from positive BCs without additional time and cost expenditure. © 2014 The Authors Clinical Microbiology and Infection © 2014 European Society of Clinical Microbiology and Infectious Diseases.

  15. Hyphenated chromatographic techniques for the rapid screening and identification of antioxidants in methanolic extracts of pharmaceutically used plants .

    NARCIS (Netherlands)

    Exarchou, V.; Fiamegos, Y.C.; Beek, van T.A.; Nanos, C.G.; Vervoort, J.J.M.

    2006-01-01

    Phytochemical analysis is an important scientific research area, which normally relies on a number of rather laborious and time-consuming techniques for compound identification. Isolation of the ingredients of plant extracts in adequate quantities for spectral and biological analysis was the basis

  16. Rapid sample preparation for detection and identification of avian influenza virus from chicken faecal samples using magnetic bead microsystem

    DEFF Research Database (Denmark)

    Dhumpa, Raghuram; Bu, Minqiang; Handberg, Kurt

    2010-01-01

    Avian influenza virus (AIV) is an infectious agent of birds and mammals. AIV is causing huge economic loss and can be a threat to human health. Reverse transcriptase polymerase chain reaction (RT-PCR) has been used as a method for the detection and identification of AIV virus. Although RT...

  17. Rapid Characterization and Identification of Flavonoids in Radix Astragali by Ultra-High-Pressure Liquid Chromatography Coupled with Linear Ion Trap-Orbitrap Mass Spectrometry.

    Science.gov (United States)

    Zhang, Jing; Xu, Xiao-Jie; Xu, Wen; Huang, Juan; Zhu, Da-yuan; Qiu, Xiao-Hui

    2015-07-01

    A simple and effective method was established for separation and characterization of flavonoid constituents in Radix Astragali (RA) by combination of ultra-high-pressure liquid chromatography with LTQ-Orbitrap tandem mass spectrometry (u-HPLC-LTQ-Orbitrap-MS(n)). For three major structural types of flavonoids, the proposed fragmentation pathways and major diagnostic fragment ions of isoflavones, pterocarpans and isoflavans were investigated to trace isoflavonoid derivatives in crude plant extracts. Based on the systematic identification strategy, 48 constituents were rapidly detected and characterized or tentatively identified, many of which were first reported in RA. The u-PHLC-LTQ-Orbitrap MS(n) platform was proved as an effective tool for rapid qualitative analysis of secondary metabolite productions from natural resources. © The Author 2014. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

  18. Identification of a Novel TECTA mutation in a Chinese DFNA8/12 family with prelingual progressive sensorineural hearing impairment.

    Directory of Open Access Journals (Sweden)

    Zhengyue Li

    Full Text Available Tectorial membrane, an extracellular matrix of the cochlea, plays a crucial role in the transmission of sound to the sensory hair cells. Alpha-tectorin is the most important noncollagenous component of the tectorial membrane and the otolith membrane in the maculae of the vestibular system. Defects in TECTA, the gene encodes alpha-tectorin, are cause of both dominant (DFNA8/12 and recessive (DFNB21 forms of deafness. Here, we report a three-generation Chinese family characterized by prelingual progressive sensorineural hearing impairment. We mapped the disease locus to chromosome 11q23-24 region, overlapping with the DFNA8/12 locus. Sequencing of candidate gene TECTA revealed a heterozygous c.5945C>A substitution in exon 19, causing amino acid substitution of Ala to Asp at a conservative position 1982. The A1982D substitution is consistent with hearing loss in this Chinese family and has not been found in 200 random control chromosomes. To our knowledge, this is the first TECTA mutation identified in Chinese population. Our data provides additional molecular and clinical information for establishing a better genotype-phenotype understanding of DFNA8/12.

  19. Identification of Epithelial-Mesenchymal Transition-related Target Genes Induced by the Mutation of Smad3 Linker Phosphorylation

    Science.gov (United States)

    Park, Sujin; Yang, Kyung-Min; Park, Yuna; Hong, Eunji; Hong, Chang Pyo; Park, Jinah; Pang, Kyoungwha; Lee, Jihee; Park, Bora; Lee, Siyoung; An, Haein; Kwak, Mi-Kyung; Kim, Junil; Kang, Jin Muk; Kim, Pyunggang; Xiao, Yang; Nie, Guangjun; Ooshima, Akira

    2018-01-01

    Background Smad3 linker phosphorylation plays essential roles in tumor progression and metastasis. We have previously reported that the mutation of Smad3 linker phosphorylation sites (Smad3-Erk/Pro-directed kinase site mutant constructs [EPSM]) markedly reduced the tumor progression while increasing the lung metastasis in breast cancer. Methods We performed high-throughput RNA-Sequencing of the human prostate cancer cell lines infected with adenoviral Smad3-EPSM to identify the genes regulated by Smad3-EPSM. Results In this study, we identified genes which are differentially regulated in the presence of Smad3-EPSM. We first confirmed that Smad3-EPSM strongly enhanced a capability of cell motility and invasiveness as well as the expression of epithelial-mesenchymal transition marker genes, CDH2, SNAI1, and ZEB1 in response to TGF-β1 in human pancreatic and prostate cancer cell lines. We identified GADD45B, CTGF, and JUNB genes in the expression profiles associated with cell motility and invasiveness induced by the Smad3-EPSM. Conclusions These results suggested that inhibition of Smad3 linker phosphorylation may enhance cell motility and invasiveness by inducing expression of GADD45B, CTGF, and JUNB genes in various cancers. PMID:29629343

  20. In Silico Screening, Alanine Mutation, and DFT Approaches for Identification of NS2B/NS3 Protease Inhibitors

    Directory of Open Access Journals (Sweden)

    R. Balajee

    2016-01-01

    Full Text Available To identify the ligand that binds to a target protein with high affinity is a nontrivial task in computer-assisted approaches. Antiviral drugs have been identified for NS2B/NS3 protease enzyme on the mechanism to cleave the viral protein using the computational tools. The consequence of the molecular docking, free energy calculations, and simulation protocols explores the better ligand. It provides in-depth structural insights with the catalytic triad of His51, Asp75, Ser135, and Gly133. The MD simulation was employed here to predict the stability of the complex. The alanine mutation has been performed and its stability was monitored by using the molecular dynamics simulation. The minimal RMSD value suggests that the derived complexes are close to equilibrium. The DFT outcome reveals that the HOMO-LUMO gap of Ligand19 is 2.86 kcal/mol. Among the considered ligands, Ligand19 shows the lowest gap and it is suggested that the HOMO of Ligand19 may transfer the electrons to the LUMO in the active regions. The calculated binding energy of Ligand19 using the DFT method is in good agreement with the docking studies. The pharmacological activity of ligand was performed and satisfies Lipinski rule of 5. Moreover, the computational results are compared with the available IC50 values of experimental results.

  1. Diagnosis of Xeroderma Pigmentosum Groups A and C by Detection of Two Prevalent Mutations in West Algerian Population: A Rapid Genotyping Tool for the Frequent XPC Mutation c.1643_1644delTG.

    Science.gov (United States)

    Bensenouci, Salima; Louhibi, Lotfi; De Verneuil, Hubert; Mahmoudi, Khadidja; Saidi-Mehtar, Nadhira

    2016-01-01

    Xeroderma pigmentosum (XP) is a rare autosomal recessive disorder. Considering that XP patients have a defect of the nucleotide excision repair (NER) pathway which enables them to repair DNA damage caused by UV light, they have an increased risk of developing skin and eyes cancers. In the present study, we investigated the involvement of the prevalent XPA and XPC genes mutations-nonsense mutation (c.682C>T, p.Arg228X) and a two-base-pair (2 bp) deletion (c.1643_1644delTG or p.Val548Ala fsX25), respectively-in 19 index cases from 19 unrelated families in the West of Algeria. For the genetic diagnosis of XPA gene, we proceeded to PCR-RFLP. For the XPC gene, we validated a routine analysis which includes a specific amplification of a short region surrounding the 2 bp deletion using a fluorescent primer and fragment sizing (GeneScan size) on a sequencing gel. Among the 19 index cases, there were 17 homozygous patients for the 2 bp deletion in the XPC gene and 2 homozygous patients carrying the nonsense XPA mutation. Finally, XPC appears to be the major disease-causing gene concerning xeroderma pigmentosum in North Africa. The use of fragment sizing is the simplest method to analyze this 2 bp deletion for the DNA samples coming from countries where the mutation c.1643_1644delTG of XPC gene is prevalent.

  2. Citrullinemia type I, classical variant. Identification of ASS-p~G390R (c.1168G>A) mutation in families of a limited geographic area of Argentina: a possible population cluster.

    Science.gov (United States)

    Laróvere, Laura E; Angaroni, Celia J; Antonozzi, Sandra L; Bezard, Miriam B; Shimohama, Mariko; de Kremer, Raquel Dodelson

    2009-07-01

    Citrullinemia type I (CTLN1) is an urea cycle defect caused by mutations in the argininosuccinate synthetase gene. We report the first identification in Argentina of patients with CTLN1 in a limited geographic area. Molecular analysis in patient/relatives included PCR, sequencing and restriction enzyme assay. The studied families showed the same mutation: ASS~p.G390R, associated with the early-onset/severe phenotype. We postulate a possible population cluster. A program to know the carrier frequency in that population is in progress.

  3. Reliable and reproducible method for rapid identification of Nocardia species by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

    Science.gov (United States)

    Toyokawa, Masahiro; Kimura, Keigo; Nishi, Isao; Sunada, Atsuko; Ueda, Akiko; Sakata, Tomomi; Asari, Seishi

    2013-01-01

    Recently, matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) has been challenged for the identification of Nocardia species. However, the standard ethanol-formic acid extraction alone is insufficient in allowing the membrane proteins of Nocardia species to be ionized by the matrix. We therefore aimed to establish our new extraction method for the MALDI-TOF MS-based identification of Nocardia species isolates. Our modified extraction procedure is through dissociation in 0.5% Tween-20 followed by bacterial heat-inactivation, mechanical breaking of the cell wall by acid-washed glass beads and protein extraction with formic acid and acetonitrile. As reference methods for species identification, full-length 16S rRNA gene sequencing and some phenotypical tests were used. In a first step, we made our own Nocardia database by analyzing 13 strains (13 different species including N. elegans, N. otitidiscaviarum, N. asiatica, N. abscessus, N. brasiliensis, N. thailandica, N. farcinica, N. nova, N. mikamii, N. cyriacigeorgica, N. asteroids, Nocardiopsis alba, and Micromonospora sp.) and registered to the MALDI BioTyper database. Then we established our database. The analysis of 12 challenge strains using the our database gave a 100% correct identification, including 8 strains identified to the species level and 4 strains to the genus level (N. elegans, N. nova, N. farcinica, Micromonospora sp.) according to the manufacture's log score specifications. In the estimation of reproducibility of our method intended for 4 strains, both within-run and between-run reproducibility were excellent. These data indicates that our method for rapid identification of Nocardia species is with reliability, reproducibility and cost effective.

  4. Universal screening for Lynch syndrome in endometrial cancers: frequency of germline mutations and identification of patients with Lynch-like syndrome.

    Science.gov (United States)

    Dillon, Jessica L; Gonzalez, Jorge L; DeMars, Leslie; Bloch, Katarzyna J; Tafe, Laura J

    2017-12-01

    Lynch syndrome (LS) is an inherited clinical syndrome characterized by a high risk of colorectal, endometrial (lifetime risk of up to 60%), ovarian, and urinary tract cancers. The diagnosis is confirmed by identification of germline mutations in the DNA mismatch repair genes MLH1, PMS2, MSH2, MSH6, or EPCAM. In 2015, our institution implemented universal screening of endometrial cancer (EC) hysterectomy specimens by mismatch repair immunohistochemistry (IHC) with reflex MLH1 promoter hypermethylation analysis for tumors with loss of MLH1/PMS2 expression. Patients with tumors negative for MLH1 methylation and those with a loss of the heterodimer pair MSH2 and MSH6, or isolated loss of either PMS2 or MSH6 were referred to the Familial Cancer Program for genetic counseling and consideration of germline testing. Between May 2015 to Dec 2016, 233 EC patients were screened by IHC for LS with a median age of 63 years. Sixty tumors (27%) had abnormal IHC staining results. Fifty-one (22%) harbored heterodimeric loss of MLH1 and PMS2, 49 of which showed MLH1 promoter methylation (1 failure, 1 negative). One showed loss of MLH1/PMS2 and MSH6, 2 showed loss of MSH2/MSH6, and 6 had isolated loss of MSH6 only. Ten patients underwent genetic counseling, and germline testing was performed in 8; LS was confirmed in 5 patients (2.1%). In addition, 3 patients with negative germline testing and presumed Lynch-like syndrome were identified and offered additional somatic testing. Universal screening for LS in EC patients has yielded positive results for identification of patients at risk for this inherited syndrome. Copyright © 2017 Elsevier Inc. All rights reserved.

  5. Identification of two new mutations in the GPR98 and the PDE6B genes segregating in a Tunisian family.

    Science.gov (United States)

    Hmani-Aifa, Mounira; Benzina, Zeineb; Zulfiqar, Fareeha; Dhouib, Houria; Shahzadi, Amber; Ghorbel, Abdelmonem; Rebaï, Ahmed; Söderkvist, Peter; Riazuddin, Sheikh; Kimberling, William J; Ayadi, Hammadi

    2009-04-01

    Autosomal recessive retinitis pigmentosa (ARRP) is a genetically heterogeneous disorder. ARRP could be associated with extraocular manifestations that define specific syndromes such as Usher syndrome (USH) characterized by retinal degeneration and congenital hearing loss (HL). The USH type II (USH2) associates RP and mild-to-moderate HL with preserved vestibular function. At least three genes USH2A, the very large G-protein-coupled receptor, GPR98, and DFNB31 are responsible for USH2 syndrome. Here, we report on the segregation of non-syndromic ARRP and USH2 syndrome in a consanguineous Tunisian family, which was previously used to define USH2B locus. With regard to the co-occurrence of these two different pathologies, clinical and genetic reanalysis of the extended family showed (i) phenotypic heterogeneity within USH2 patients and (ii) excluded linkage to USH2B locus. Indeed, linkage analysis disclosed the cosegregation of the USH2 phenotype with the USH2C locus markers, D5S428 and D5S618, whereas the ARRP perfectly segregates with PDE6B flanking markers D4S3360 and D4S2930. Molecular analysis revealed two new missense mutations, p.Y6044C and p.W807R, occurring in GPR98 and PDE6B genes, respectively. In conclusion, our results show that the USH2B locus at chromosome 3p23-24.2 does not exist, and we therefore withdraw this locus designation. The combination of molecular findings for GPR98 and PDE6B genes enable us to explain the phenotypic heterogeneity and particularly the severe ocular affection first observed in one USH2 patient. This report presents an illustration of how consanguinity could increase familial clustering of multiple hereditary diseases within the same family.

  6. Rapid identification of pork for halal authentication using the electronic nose and gas chromatography mass spectrometer with headspace analyzer.

    Science.gov (United States)

    Nurjuliana, M; Che Man, Y B; Mat Hashim, D; Mohamed, A K S

    2011-08-01

    The volatile compounds of pork, other meats and meat products were studied using an electronic nose and gas chromatography mass spectrometer with headspace analyzer (GCMS-HS) for halal verification. The zNose™ was successfully employed for identification and differentiation of pork and pork sausages from beef, mutton and chicken meats and sausages which were achieved using a visual odor pattern called VaporPrint™, derived from the frequency of the surface acoustic wave (SAW) detector of the electronic nose. GCMS-HS was employed to separate and analyze the headspace gasses from samples into peaks corresponding to individual compounds for the purpose of identification. Principal component analysis (PCA) was applied for data interpretation. Analysis by PCA was able to cluster and discriminate pork from other types of meats and sausages. It was shown that PCA could provide a good separation of the samples with 67% of the total variance accounted by PC1. Copyright © 2011 Elsevier Ltd. All rights reserved.

  7. A DNA Barcode-Based RPA Assay (BAR-RPA) for Rapid Identification of the Dry Root of Ficus hirta (Wuzhimaotao).

    Science.gov (United States)

    Tian, Enwei; Liu, Qianqian; Ye, Haoting; Li, Fang; Chao, Zhi

    2017-12-18

    Background: Wuzhimaotao (the dry root of Ficus hirta ) is used as both medicine and food ingredient by the locals in areas around Nanling Mountains of China. Due to its very similar external morphologies with Duanchangcao (the root of Gelsemium elegans , which contains gelsemine that is extremely neurotoxic) and the associated growth of these two plants, incidents of food poisoning and even death frequently occur, resulting from the misuse of Duanchangcao as Wuzhimaotao. The aim of this study is to develop a fast, even, on-spot approach to identification of Wuzhimaotao. Methods: We used DNA barcode-based recombinase polymerase amplification (BAR-RPA) with species-specific primers targeting the internal transcribed spacer (ITS) region of the rDNA of F. hirta. BAR-RPA reaction time and temperature were optimized and the specificity and sensitivity of BAR-RPA species-specific primers were assessed. Results: This technique showed a high specificity and sensitivity to amplify the genomic DNA of F. hirta and allowed for rapid amplification (within 15 min) of the ITS region under a constant and mild temperature range of 37-42 °C without using thermocyclers. Conclusions: The BAR-RPA assay with a fast DNA extraction protocol provides a simple, energy-saving, and rapid method for identification of Wuzhimaotao in both laboratory and field settings.

  8. Accurate, safe, and rapid method of intraoperative tumor identification for totally laparoscopic distal gastrectomy: injection of mixed fluid of sodium hyaluronate and patent blue.

    Science.gov (United States)

    Nakagawa, Masatoshi; Ehara, Kazuhisa; Ueno, Masaki; Tanaka, Tsuyoshi; Kaida, Sachiko; Udagawa, Harushi

    2014-04-01

    In totally laparoscopic distal gastrectomy, determining the resection line with safe proximal margins is often difficult, particularly for tumors located in a relatively upper area. This is because, in contrast to open surgery, identifying lesions by palpating or opening the stomach is essentially impossible. This study introduces a useful method of tumor identification that is accurate, safe, and rapid. On the operation day, after inducing general anesthesia, a mixture of sodium hyaluronate and patent blue is injected into the submucosal layer of the proximal margin. When resecting stomach, all marker spots should be on the resected side. In all cases, the proximal margin is examined histologically by using frozen sections during the operation. From October 2009 to September 2011, a prospective study that evaluated this method was performed. A total of 34 patients who underwent totally laparoscopic distal gastrectomy were enrolled in this study. Approximately 5 min was required to complete the procedure. Proximal margins were negative in all cases, and the mean ± standard deviation length of the proximal margin was 23.5 ± 12.8 mm. No side effects, such as allergy, were encountered. As a method of tumor identification for totally laparoscopic distal gastrectomy, this procedure appears accurate, safe, and rapid.

  9. A DNA Barcode-Based RPA Assay (BAR-RPA for Rapid Identification of the Dry Root of Ficus hirta (Wuzhimaotao

    Directory of Open Access Journals (Sweden)

    Enwei Tian

    2017-12-01

    Full Text Available Background: Wuzhimaotao (the dry root of Ficus hirta is used as both medicine and food ingredient by the locals in areas around Nanling Mountains of China. Due to its very similar external morphologies with Duanchangcao (the root of Gelsemium elegans, which contains gelsemine that is extremely neurotoxic and the associated growth of these two plants, incidents of food poisoning and even death frequently occur, resulting from the misuse of Duanchangcao as Wuzhimaotao. The aim of this study is to develop a fast, even, on-spot approach to identification of Wuzhimaotao. Methods: We used DNA barcode-based recombinase polymerase amplification (BAR-RPA with species–specific primers targeting the internal transcribed spacer (ITS region of the rDNA of F. hirta. BAR-RPA reaction time and temperature were optimized and the specificity and sensitivity of BAR-RPA species–specific primers were assessed. Results: This technique showed a high specificity and sensitivity to amplify the genomic DNA of F. hirta and allowed for rapid amplification (within 15 min of the ITS region under a constant and mild temperature range of 37–42 °C without using thermocyclers. Conclusions: The BAR-RPA assay with a fast DNA extraction protocol provides a simple, energy-saving, and rapid method for identification of Wuzhimaotao in both laboratory and field settings.

  10. Four-channel asymmetric Real-Time PCR hybridization probe assay: a rapid pre-screening method for critical BCR-ABL kinase domain mutations.

    Science.gov (United States)

    Martinez-Serra, Jordi; Gutiérrez, Antonio; Marcús, Toni F; Soverini, Simona; Amat, Juan Carlos; Navarro-Palou, María; Ros, Teresa; Bex, Teresa; Ballester, Carmen; Bauça, Josep Miquel; SanFelix, Sara; Novo, Andrés; Vidal, Carmen; Santos, Carmen; Besalduch, Joan

    2012-03-01

    Within the laboratory protocols, used for the study of BCR-ABL resistance mutations in chronic myeloid leukemia patients treated with Imatinib, direct sequencing remains the reference method. Since the incidence of patients with a mutation-related loss of response is not very high, it is very useful in the routine laboratory to perform a fast pre-screening method. With this in mind, we have designed a new technique, based on a single Real-Time FRET-based PCR, followed by a study of melting peaks. This new tool, developed in a LightCycler 2.0, combines four different fluorescence channels for the simultaneous detection, in a single close tube, of critical mutations within the ABL kinase domain. Assay evaluation performed on 33 samples, previously genotyped by sequentiation, resulted in full concordance of results. This new methodology detects in a few steps the presence of critical mutations associated to Imatinib resistance. Copyright © 2012 Elsevier Inc. All rights reserved.

  11. Rapid characterization of disease-causing mutations in the low density lipoprotein receptor (LDL-R) gene by overexpression in COS cells

    DEFF Research Database (Denmark)

    Jensen, T G; Andresen, B S; Jensen, H K

    1996-01-01

    To characterize disease-causing mutations in the low density lipoprotein receptor (LDL-R) gene, COS cells are transfected with the mutant gene in an EBV-based expression vector and characterized by flow cytometry. Using antibodies against the LDL-receptor the amount of receptor protein on the cel...

  12. Disparity mutagenesis model possesses the ability to realize both stable and rapid evolution in response to changing environments without altering mutation rates.

    Directory of Open Access Journals (Sweden)

    Ichiro Fujihara

    2016-08-01

    As long as the fidelity difference between the lagging and leading strand was kept high enough, the robustness of the disparity model was very high. The acceleration or slowdown of evolution can be unambiguously introduced only by environmental changes, and the seesawing mutation rate is not the necessary condition for changing the speed of evolution.

  13. Expediting the Analysis of Qualitative Data in Evaluation: A Procedure for the Rapid Identification of Themes from Audio Recordings (RITA)

    Science.gov (United States)

    Neal, Jennifer Watling; Neal, Zachary P.; VanDyke, Erika; Kornbluh, Mariah

    2015-01-01

    Qualitative data offer advantages to evaluators, including rich information about stakeholders' perspectives and experiences. However, qualitative data analysis is labor-intensive and slow, conflicting with evaluators' needs to provide punctual feedback to their clients. In this method note, we contribute to the literature on rapid evaluation and…

  14. Rapid-resolution HPLC with specgtrometric detection for the determination and identification of isoflavones in soy preparations and plant extracts

    Czech Academy of Sciences Publication Activity Database

    Klejdus, B.; Vacek, J.; Benešová, L.; Kopecký, Jiří; Lapčík, O.; Kubáň, V.

    2007-01-01

    Roč. 389, - (2007), s. 2277-2285 ISSN 1618-2642 R&D Projects: GA ČR GA525/07/0338; GA ČR GA525/06/0864 Institutional research plan: CEZ:AV0Z50200510 Keywords : rapid resolution * fast chromatography * hplc Subject RIV: EE - Microbiology, Virology Impact factor: 2.867, year: 2007

  15. Potential of MALDI-TOF mass spectrometry as a rapid detection technique in plant pathology: identification of plant-associated microorganisms.

    Science.gov (United States)

    Ahmad, Faheem; Babalola, Olubukola O; Tak, Hamid I

    2012-09-01

    Plant diseases caused by plant pathogens substantially reduce crop production every year, resulting in massive economic losses throughout the world. Accurate detection and identification of plant pathogens is fundamental to plant pathogen diagnostics and, thus, plant disease management. Diagnostics and disease-management strategies require techniques to enable simultaneous detection and quantification of a wide range of pathogenic and non-pathogenic microorganisms. Over the past decade, rapid development of matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) techniques for characterization of microorganisms has enabled substantially improved detection and identification of microorganisms. In the biological sciences, MALDI-TOF MS is used to analyze specific peptides or proteins directly desorbed from intact bacteria, fungal spores, nematodes, and other microorganisms. The ability to record biomarker ions, in a broad m/z range, which are unique to and representative of individual microorganisms, forms the basis of taxonomic identification of microorganisms by MALDI-TOF MS. Recent advances in mass spectrometry have initiated new research, i.e. analysis of more complex microbial communities. Such studies are just beginning but have great potential for elucidation not only of the interactions between microorganisms and their host plants but also those among different microbial taxa living in association with plants. There has been a recent effort by the mass spectrometry community to make data from large scale mass spectrometry experiments publicly available in the form of a centralized repository. Such a resource could enable the use of MALDI-TOF MS as a universal technique for detection of plant pathogens and non-pathogens. The effects of experimental conditions are sufficiently understood, reproducible spectra can be obtained from computational database search, and microorganisms can be rapidly characterized by genus, species

  16. High resolution melting curve analysis as a new tool for rapid identification of canine parvovirus type 2 strains.

    Science.gov (United States)

    Bingga, Gali; Liu, Zhicheng; Zhang, Jianfeng; Zhu, Yujun; Lin, Lifeng; Ding, Shuangyang; Guo, Pengju

    2014-01-01

    A high resolution melting (HRM) curve method was developed to identify canine parvovirus type 2 (CPV-2) strains by nested PCR. Two sets of primers, CPV-426F/426R and CPV-87R/87F, were designed that amplified a 52 bp and 53 bp product from the viral VP2 capsid gene. The region amplified by CPV-426F/426R included the A4062G and T4064A mutations in CPV-2a, CPV-2b and CPV-2c. The region amplified by CPV-87F/87R included the A3045T mutation in the vaccine strains of CPV-2 and CPV-2a, CPV-2b and CPV-2c. Faecal samples were obtained from 30 dogs that were CPV antigen-positive. The DNA was isolated from the faecal samples and PCR-amplified using the two sets of primers, and genotyped by HRM curve analysis. The PCR-HRM assay was able to distinguish single nucleotide polymorphisms between CPV-2a, CPV-2b and CPV-2c using CPV-426F/426R. CPV-2a was distinguished from CPV-2b and CPV-2c by differences in the melting temperature. CPV-2b and CPV-2c could be distinguished based on the shape of the melting curve after generating heteroduplexes using a CPV-2b reference sample. The vaccine strains of CPV-2 were identified using CPV-87F/87R. Conventional methods for genotyping CPV strains are labor intensive, expensive or time consuming; the present PCR-based HRM assay might be an attractive alternative. Copyright © 2014 Elsevier Ltd. All rights reserved.

  17. Identification of a c.544C>T mutation in WDR34 as a deleterious recessive allele of short rib-polydactyly syndrome

    Directory of Open Access Journals (Sweden)

    Shu-Han You

    2017-12-01

    Conclusion: This study was the first to identify c.544C > T [p.Arg182Trp] mutation in WDR34 in a patient with SRPS. According to the database, the homozygous mutation of c.544C > T in WDR34 was deleterious and the prevalence of heterozygous mutation was relatively higher in Asian population. More studies of this mutation in patients with SRPS are required.