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Effective Detection (effective + detection)
Selected AbstractsEffective detection of corrected dystrophin loci in mdx mouse myogenic precursors,HUMAN MUTATION, Issue 8 2007Marian Todaro Abstract Targeted corrective gene conversion (TCGC) holds much promise as a future therapy for many hereditary diseases in humans. Mutation correction frequencies varying between 0.0001% and 40% have been reported using chimeraplasty, oligoplasty, triplex-forming oligonucleotides, and small corrective PCR amplicons (CPA). However, PCR technologies used to detect correction events risk either falsely indicating or greatly exaggerating the presence of corrected loci. This is a problem that is considerably exacerbated by attempted improvement of the TCGC system using high corrective nucleic acid (CNA) to nuclear ratios. Small fragment homologous replacement (SFHR)-mediated correction of the exon 23 dystrophin (DMD) gene mutation in the mdx mouse model of DMD has been used in this study to evaluate the effect of increasing CPA amounts. In these experiments, we detected extremely high levels of apparently corrected loci and determined that at higher CNA to nuclear ratios the extent of locus correction was highly exaggerated by residual CNA species in the nucleic acids extracted from the treated cells. This study describes a generic locus-specific detection protocol designed to eradicate residual CNA species and avoid the artifactual or exaggerated detection of gene correction. Hum Mutat 28(8), 816,823, 2007. © 2007 Wiley-Liss, Inc. [source] Effective detection of peptides containing cysteine sulfonic acid using matrix-assisted laser desorption/ionization and laser desorption/ionization on porous silicon mass spectrometryJOURNAL OF MASS SPECTROMETRY (INCORP BIOLOGICAL MASS SPECTROMETRY), Issue 1 2006Tomoya Kinumi Abstract Cysteine sulfonic acid-containing peptides, being typical acidic peptides, exhibit low response in matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. In this study, matrix conditions and the effect of diammonium hydrogencitrate (DAHC) as additive were investigated for ionization of cysteine sulfonic acid-containing peptides in MALDI. A matrix-free ionization method, desorption/ionization on porous silicon (DIOS), was also utilized to evaluate the effect of DAHC. When equimolar three-component mixtures of peptides carrying free cysteine, cysteine sulfonic acid, and carbamidomethyl cysteine were measured by MALDI using a common matrix, ,-cyano-4-hydroxycinnamic acid (CHCA), no signal corresponding to cysteine sulfonic acid-containing peptide could be observed in the mass spectrum. However, by addition of DAHC to CHCA, the peaks of cysteine sulfonic acid-containing peptides were successfully observed, as well as when using 2,4,6-trihydroxyacetophenone (THAP) and 2,6-dihydroxyacetophenone with DAHC. In the DIOS mass spectra of these analytes, the use of DAHC also enhanced the peak intensity of the cysteine sulfonic acid-containing peptides. On the basis of studies with these model peptides, tryptic digests of oxidized peroxiredoxin 6 were examined as a complex peptide mixture by MALDI and DIOS. In MALDI, the peaks of cysteine sulfonic acid-containing peptides were observed when using THAP/DAHC as the matrix, but this was not so with CHCA. In DIOS, the signal from cysteine sulfonic acid-containing peptides was suppressed; however, the use of DAHC significantly enhanced the signal intensity with an increase in the number of observed peptides and increased signal-to-noise ratio in the DIOS spectra. The results show that DAHC in the matrix or on the DIOS chip decreases discrimination and suppression effects in addition to suppressing alkali-adduct ions, which leads to a beneficial effect on protonation of peptides containing cysteine sulfonic acid. Copyright © 2005 John Wiley & Sons, Ltd. [source] Development and evaluation of a one-step loop-mediated isothermal amplification for detection of spring viraemia of carp virusJOURNAL OF APPLIED MICROBIOLOGY, Issue 4 2008Z. Liu Abstract Aim:, Spring viraemia of carp virus (SVCV) is the causative agent of SVC disease. The main aim of our study was to develop a one-step reverse transcription loop-mediated isothermal amplification (RT-LAMP) assay for rapid, sensitive and effective detection of SVCV. Methods and Results:, A set of four specific primers, two outer and two inner primers were designed based on the SVCV M gene for RT-LAMP assay. The sensitivity and specificity of RT-LAMP were determined and clinical test was performed under optimized amplification conditions (64°C, 60 min). The results showed that the assay has a high specificity and the detection limit was 80 copies using 10-fold series dilutions of SVCV RNA, 10 times more sensitive than nest reverse transcription-polymerase chain reaction. In the detection of 472 fish samples, this assay showed excellent agreement with the standard virus isolation method (, = 0·807). Conclusions:, A sensitive and specific RT-LAMP assay was successfully developed to monitor and detect SVCV. Significance and Impact of the Study:, This work provides a robust method for evaluating the risk of SVCV. Given the advantages of LAMP in the detection of SVCV, this method can be applied to diagnose other viruses, which pose serious threats to the aquaculture industry. [source] Rapid and effective detection of anthrax spores in soil by PCRJOURNAL OF APPLIED MICROBIOLOGY, Issue 4 2003H.I. Cheun Abstract Aims: To detect Bacillus anthracis DNA from soil using rapid and simple procedures. Methods and Results: Various amounts of B. anthracis Pasteur II spores were added artificially to 1 g of soil, which was then washed with ethanol and sterile water. Enrichment of the samples in trypticase soy broth was performed twice. A DNA template was prepared from the second enrichment culture using a FastPrep instrument. The template was then used for nested and real-time polymerase chain reaction (PCR) with B. anthracis -specific primers, to confirm the presence of B. anthracis chromosomal DNA and the pXO1/pXO2 plasmids. Conclusions: One cell of B. anthracis in 1 g of soil could be detected by nested and real-time PCR. The usefulness of the PCR method using field samples was also confirmed. Significance and Impact of the Study: The results indicate that this could be a useful method for detecting anthrax-spore contaminated soil with high sensitivity. Its application could have great impact on the progress of epidemiological surveillance. [source] The Alcohol Use Disorders Identification Test: An Update of Research FindingsALCOHOLISM, Issue 2 2007Duane F. Reinert Background: The Alcohol Use Disorders Identification Test (AUDIT) has been extensively researched to determine its capability to accurately and practically screen for alcohol problems. Methods: During the 5 years since our previous review of the literature, a large number of additional studies have been published on the AUDIT, abbreviated versions of it, its psychometric properties, and the applicability of the AUDIT for a diverse array of populations. The current article summarizes new findings and integrates them with results of previous research. It also suggests some issues that we believe are particularly in need of further study. Results: A growing body of research evidence supports the criterion validity of English version of the AUDIT as a screen for alcohol dependence as well as for less severe alcohol problems. Nevertheless, the cut-points for effective detection of hazardous drinking as well as identification of alcohol dependence or harmful use in women need to be lowered from the originally recommended value of 8 points. The AUDIT-C, the most popular short version of the AUDIT consisting solely of its 3 consumption items, is approximately equal in accuracy to the full AUDIT. Psychometric properties of the AUDIT, such as test,retest reliability and internal consistency, are quite favorable. Continued research is urged to establish the psychometric properties of non-English versions of the AUDIT, use of the AUDIT with adolescents and with older adults, and selective inclusion of alcohol biomarkers with the AUDIT in some instances. Conclusions: Research continues to support use of the AUDIT as a means of screening for the spectrum of alcohol use disorders in various settings and with diverse populations. [source] Glass-Transition Temperature (Tg) of Free-Radically Prepared Polyacrylonitrile by Inverse Gas Chromatography, 1.MACROMOLECULAR CHEMISTRY AND PHYSICS, Issue 17 2002A Study on Tg of Atactic Monodisperse Polystyrenes Abstract The glass-transition temperature (Tg) of several atactic monodisperse polystyrenes (PSt)s was studied by an inverse gas chromatographic (IGC) analysis, as a preliminary study for the detection of the Tg of polyacrylonitrile. The Tg of PSt with different molecular weights was clearly obtained by this method. The agreement in the Tg values obtained by IGC and by differential scanning calorimetry (DSC) was quite good within the molecular-weight range Mp,=,7,600,2,430,000 (where, Mp,=,(Mn,×,Mw)1/2). This method has a wide universality for the detection of the Tg of polymers. Several essential factors were extracted for the establishment of the optimum conditions for IGC measurements, such as the concentration of polymer toward a clay matrix (10 wt.-%), and the type of lower probe, decane. The best probe was determined empirically from the most effective detection in the IGC measurements of PSt. Molecular-weight dependence of the Tg of PSt. Experimental data obtained here are indicated by an arrow. Other data was taken from ref.21 [source] | ||||||||||