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Rapid Detection (rapid + detection)
Selected AbstractsDirect and Rapid Detection of Diphtherotoxin via Potentiometric Immunosensor Based on Nanoparticles Mixture and Polyvinyl Butyral as MatrixesELECTROANALYSIS, Issue 24 2005Dianping Tang Abstract In this paper a novel potentiometric immunosensor for direct and rapid detection of diphtherotoxin (D-Ag) has been developed by means of self-assembly of monoclonal diphtheria antibody (D-Ab) onto a platinum electrode based on nanoparticles mixture (containing gold nanoparticles and silica nanoparticles) and polyvinyl butyral (PVB) as matrixes. At first, D-Ab was absorbed onto the surface of nanoparticles mixture, and then they were entrapped into polyvinyl butyral sol-gel network on a platinum electrode. The detection is based on the change in the potentiometric response before and after the antigen-antibody reaction in a phosphate buffer solution (pH,7.0). The immobilized D-Ab exhibited direct potentiometric response toward D-Ag. In comparison to the conventional applied methods, this strategy could allow antibodies immobilized with higher loading amount and better retained immunoactivity, as demonstrated by potentiometric response, cyclic voltammetry and electrochemical impedance spectroscopy of the immunosensor. The immunosensor with nanoparticles mixture exhibited much higher sensitivity, better reproducibility, and long-term stability than that with gold nanoparticles or silica nanoparticles alone. The linear range was from 5.0×10,3 to 1.2,,g,mL,1 with a detection limit of 1.1×10,3,,g,mL,1. Up to 16 successive assay cycles with retentive sensitivity were achieved for the probes regenerated with in 0.2,mol,L,1 glycine-hydrochloric acid (Gly-HCl) buffer solution and 0.25,mol,L,1 NaCl. Moreover, the immunosensor with nanoparticles mixture was applied to evaluate a number of practical specimens with potentiometric results in acceptable agreement with those given by the ELISA method, implying a promising alternative approach for detecting diphtherotoxin in the clinical diagnosis. [source] Construction and Characterization of Porous SiO2/Hydrogel Hybrids as Optical Biosensors for Rapid Detection of BacteriaADVANCED FUNCTIONAL MATERIALS, Issue 14 2010Naama Massad-Ivanir Abstract The use of a new class of hybrid nanomaterials as label-free optical biosensors for bacteria detection (E. coli K12 as a model system) is demonstrated. The hybrids combine a porous SiO2 (PSiO2) optical nanostructure (a Fabry,Pérot thin film) used as the optical transducer element and a hydrogel. The hydrogel, polyacrylamide, is synthesized in situ within the nanostructure inorganic host and conjugated with specific monoclonal antibodies (IgGs) to provide the active component of the biosensor. The immobilization of the IgGs onto the hydrogel via a biotin-streptavidin system is confirmed by fluorescent labeling experiments and reflective interferometric Fourier transform spectroscopy (RIFTS). Additionally, the immobilized IgGs maintain their immunoactivity and specificity when attached to the sensor surface. Exposure of these modified-hybrids to the target bacteria results in "direct cell capture" onto the biosensor surface. These specific binding events induce predictable changes in the thin-film optical interference spectrum of the hybrid. Preliminary studies demonstrate the applicability of these biosensors for the detection of low bacterial concentrations in the range of 103,105 cell mL,1 within minutes. [source] Combining Fluorescent Probes and Biofunctional Magnetic Nanoparticles for Rapid Detection of Bacteria in Human Blood,ADVANCED MATERIALS, Issue 23 2006J. Gao Detection of bacteria in human blood within two hours is achieved through the use of vancomycin-functionalized FePt nanoparticles in combination with a vancomycin-conjugated fluorescent probe, as shown in the figure (B: bacteria; Van: vancomycin; FLA: fluorescein amine). This promises to be a sensitive and rapid protocol for detecting bacteria in blood products or other targets in biological samples. [source] Iridium Oxide Film-Enhanced Impedance Immunosensor for Rapid Detection of Carcinoembyronic AntigenCHINESE JOURNAL OF CHEMISTRY, Issue 9 2007Yan-Jun Ding Abstract A simple, rapid and sensitive impedance immunosensor based on iridium oxide (IrOx) thin film for the detection of carcinoembyronic antigen (CEA) in human sera has been proposed. Gold electrode was electrochemically modified with IrOx thin film and simultaneously functionalized with protein A (PA) to bind anti-CEA antibodies in an orientated way. It has been found that the antibody loading amount was dependent on the PA concentration and the deposition time of IrOx matrix. Under the optimized experimental conditions, the electron transfer resistances obtained were linearly related to the CEA concentration ranging from 36.2 to 460.0 ng/mL, with a detection limit of 28.0 ng/mL. Analytical results of clinical samples from cancer patients show that the proposed immunoassay is reasonably comparable with the chemiluminescence immunoassay (CLIA), indicating the feasibility of using the proposed method for CEA immunoassay in clinical laboratory. [source] Rapid detection and identification of counterfeit of adulterated products of synthetic phosphodiesterase type-5 inhibitors with an atmospheric solids analysis probeDRUG TESTING AND ANALYSIS, Issue 2 2010Marian Twohig Abstract The market success of the three approved synthetic phosphodiesterase type-5 (PDE-5) inhibitors for the treatment of erectile dysfunction has led to an explosion in counterfeit versions of these drugs. In parallel a large market has developed for herbal products claimed to be natural alternatives to these synthetic drugs. The herbal products are heavily advertised on the internet and are freely available to purchase without prescription. Furthermore, adulteration of these supposed natural medicines is a very common and serious phenomenon. Recent reports have shown that the adulteration has extended to the analogues of the three approved synthetic PDE-5 inhibitors. An Atmospheric Solids Analysis Probe (ASAP) was used for the direct analysis of the counterfeit pharmaceuticals and herbal products. Using the ASAP combined with time-of-flight mass spectrometry (TOF MS) it was possible to detect fraudulent counterfeit tablets. The physical appearance of the pills resembled the pills from the original manufacturer but contained the wrong active pharmaceutical ingredient (API). Detecting adulteration for five herbal supplements marketed as natural alternatives to PDE-5 inhibitors was also possible using the ASAP. Three types of adulteration were found in the five samples: adulteration with tadalafil or sildenafil, mixed adulteration (tadalafil and sildenafil), and adulteration with analogues of these drugs. Copyright © 2010 John Wiley & Sons, Ltd. [source] Rapid detection of Staphylococcus aureus by a combination of monoclonal antibody-coated latex and capillary electrophoresisELECTROPHORESIS, Issue 9 2006Peng Gao Abstract The rapid detection of pathogenic bacteria is extremely important in biotechnology and clinical diagnosis. CE has been utilized in the field of bacterial analysis for many years, but to some extent, simultaneous separation and identification of certain microbes from complex samples by CE coupled with UV detector is still a challenge. In this paper, we propose a new strategy for rapid separation and identification of Staphylococcus aureus (S.,aureus) in bacterial mixtures by means of specific mAb-coated latex coupled with CZE. An appropriate set of conditions that selectively isolated S.,aureus from the microorganisms Escherichia coli, Pseudomonas aeruginosa, and Klebsiella pneumoniae were established. S.,aureus could be differentiated from the others by unique peaks in the electropherograms. The validity was also confirmed by LIF with antibodies specific to both the latex and the microbial cells. The LOD is as low as 9.0×105 colony forming unit/mL. We have also utilized this technology to identify S.,aureus in a stool sample coming from a healthy volunteer spiked successfully with S.,aureus. This CZE-UV technique can be applied to rapid diagnosis of enteritis caused by S.,aureus or other bacterial control-related fields needing rapid identification of target pathogens from microbial mixtures. In theory, this method is suitable for the detection of any bacterium as long as corresponding bacterium-specific antibody-coated latex is available. [source] Rapid detection of yeast rRNA genes with primed in situ (PRINS) labelingFEMS YEAST RESEARCH, Issue 4 2009Maciej Wnuk Abstract In yeast, rRNA genes can be detected with the FISH technique using rRNA gene probes. This technique yields reliable, reproducible and precise results, but is time-consuming. Here, the primed in situ DNA synthesis (PRINS) procedure has been optimized for rapid detection of yeast rRNA genes. PRINS, which is as sensitive as PCR and allows cytological localization of analyzed sequences, can be adapted for various screening tests requiring fast labeling of rRNA genes. [source] Rapid detection of fungal endophytes in grasses for large-scale studiesFUNCTIONAL ECOLOGY, Issue 4 2006S. KOH Summary 1Standard visual screening methods for determining the qualitative and quantitative presence of fungal endophytes are too time-consuming for large-scale ecological studies. 2We investigated whether commercially available immunoblot kits, using monoclonal antibody techniques and designed for rapid-screening of the presence of Neotyphodium endophytes in fresh samples of the pasture grasses Festuca arundinacea and Lolium perenne, could be used for Neotyphodium detection using other grasses and preserved samples. We also determined whether immunoblot kits could provide quantitative information about the amount of Neotyphodium in the grass. 3The kits accurately detected endophyte presence in F. rubra, F. ovina, F. pratensis and F. altaica, in both preserved samples (dried and fixed), including 12-year-old stored, dried samples of F. rubra. 4Endophytes were detected in 7-day-old seedlings of Lolium perenne, 3 days (30%) earlier than previously recognized. 5The intensity of the coloured tissue prints on scanned immunoblot cards was significantly positively correlated with hyphal density, demonstrating a previously unrecognized accurate quantitative application. 6These findings greatly reduce logistical barriers to large-scale field research into the broader ecological significance of Neotyphodium in temperate and arctic grasses in non-agricultural ecosystems (particularly in remote areas) and suggest potential for estimating historical infection rates using stored and herbarium specimens. [source] Rapid detection of methylation change at H19 in human imprinting disorders using methylation-sensitive high-resolution melting,HUMAN MUTATION, Issue 10 2008Tomasz K. Wojdacz Abstract Beckwith Wiedemann syndrome (BWS) and Russell Silver syndrome (RS) are growth disorders with opposing epimutations affecting the H19/IGF2 imprinting center at 11p15.5. Overgrowth and tumor risk in BWS is caused by aberrant expression of the paternally expressed, imprinted IGF2 gene, occurring as a consequence of mosaic hypermethylation within the imprinting center, or to mosaic paternal uniparental disomy (UPD). RS is characterized by severe intrauterine growth retardation (IUGR). A subset of RS cases were recently shown to have mosaic hypomethylation within the H19/IGF2 imprinting center, predicted to silence paternally expressed IGF2 in early development. Molecular diagnosis for BWS and RS involves methylation analysis of the H19 locus, enabling discrimination of allelic methylation patterns. In this study, methylation-sensitive high-resolution melting analysis (MS-HRM) was used to analyze methylation within the intergenic region of the H19 locus. A total of 36 samples comprising normal control (11), BWS (19), and RS (six) DNA were analyzed in a blinded study and scored as hypermethylated, normal, or hypomethylated. Results were compared with those derived by methylation-sensitive Southern blotting using the restriction enzymes Rsa I and Hpa II. A total of 100% concordance was obtained for the Southern blotting and MS-HRM scores. A total of three samples with paternal duplication affecting the H19/IGF2 region were scored as equivocal by both methods; however, 33 out of 36 (92%) the samples were unambiguously scored as being hypermethylated, hypomethylated, or normally methylated using MS-HRM. We conclude that MS-HRM is a rapid, cost-effective, and sensitive method for screening mosaic methylation changes at the H19 locus in BWS and RS. Hum Mutat 0,1,6, 2008. © 2008 Wiley-Liss, Inc. [source] Rapid detection of submicroscopic chromosomal rearrangements in children with multiple congenital anomalies using high density oligonucleotide arrays,,HUMAN MUTATION, Issue 5 2006Jeffrey E. Ming Abstract Chromosomal rearrangements such as microdeletions and interstitial duplications are the underlying cause of many human genetic disorders. These disorders can manifest in the form of multiple congenital anomalies (MCA), which are a significant cause of morbidity and mortality in children. The major limitations of cytogenetic tests currently used for the detection of such chromosomal rearrangements are low resolution and limited coverage of the genome. Thus, it is likely that children with MCA may have submicroscopic chromosomal rearrangements that are not detectable by current techniques. We report the use of a commercially available, oligonucleotide-based microarray for genome-wide analysis of copy number alterations. First, we validated the microarray in patients with known chromosomal rearrangements. Next, we identified previously undetected, de novo chromosomal deletions in patients with MCA who have had a normal high-resolution karyotype and subtelomeric fluorescence in situ hybridization (FISH) analysis. These findings indicate that high-density, oligonucleotide-based microarrays can be successfully used as tools for the detection of chromosomal rearrangement in clinical samples. Their higher resolution and commercial availability make this type of microarray highly desirable for application in the diagnosis of patients with multiple congenital defects. Hum Mutat 27(5), 467,473, 2006. © Published 2006 Wiley-Liss, Inc. [source] Rapid detection of metastasis of gastric cancer using reverse transcription loop-mediated isothermal amplificationINTERNATIONAL JOURNAL OF CANCER, Issue 5 2007Daisuke Horibe Abstract Tailor-made surgeries for patients with solid malignancies have been under consideration on the basis of the development of new approaches for minor metastatic foci of malignant tumors. Accurate and reliable methods to detect metastases in biopsy specimens with certain rapidity are essential for the performance of these surgeries. The aim of this study was to develop a rapid and practical method to detect metastasis in specimens from patients with gastric carcinoma with the use of reverse transcription loop-mediated isothermal amplification (RT-LAMP) reaction, a novel technique for detecting mRNA expressions of targeted sequences with high sensitivity, specificity and rapidity under isothermal conditions. RT-LAMP primers to detect cytokeratin19 (CK19) mRNA were generated and 92 lymph nodes (LNs) obtained from 9 patients with gastric cancer were tested for tumor metastases with this technique. Among 92 LNs, 15 were metastasis-positive by routine histopathological examination. RT-LAMP reaction detected CK19 expression in all of the pathologically positive LNs and in 16 of 77 negative LNs. Nested RT-PCR assay for CK19 expression was also performed on 2 of the 9 cases including 32 LNs. The agreement rate of CK19 expression detection by RT-LAMP and RT-PCR analysis was 31/32 (97%). The RT-LAMP technique showed similar sensitivity to detect metastases as nested RT-PCR assay, with a rapidity comparable to that of intraoperative histopathological examination with frozen sectioning and hematoxylin and eosin staining. This method is expected to play an essential role in the performance of tailor-made surgeries in the near future. © 2006 Wiley-Liss, Inc. [source] Rapid detection of bordetella pertussis by real-time PCR using SYBR green I and a LightCycler instrumentJOURNAL OF CLINICAL LABORATORY ANALYSIS, Issue 5 2004S. K. Poddar Abstract A polymerase chain reaction (PCR) assay in real-time for detection of B. pertussis using SYBR green I as the reporter fluorophore and LightCycler instrument (a thermocycler coupled to a fluorescence detection device) was established and evaluated. The amplified amplicon using series diluted control prototype strain (ATCC strain #9797) of B. pertussis was analyzed for the fluorescent melting profile, and melting temperature (Tm) was determined. When examined, amplicons using a representative set of clinical isolates of B. pertussis were found to have the same Tm value (86 ± 0.5°C, the specificity parameter of detection) as the control prototype strain as expected. Amplified product was also analyzed and detected by agarose gel electrophoresis. The detection limit by fluorescent profile and Tm analysis was 10-fold better than that detected by agarose gel analysis. J. Clin. Lab. Anal. 18:265,270, 2004. © 2004 Wiley-Liss, Inc. [source] A highly informative, multiplexed assay for the indirect detection of hemophilia A using five-linked microsatellitesJOURNAL OF THROMBOSIS AND HAEMOSTASIS, Issue 3 2006J. R. HARRAWAY Summary.,Background:,Hemophilia A is a severe bleeding disorder caused by almost 1000 different known mutations in the F8C gene. Direct mutation analysis is sometimes difficult for this disorder. When a mutation cannot be found, linkage analysis can be used for prenatal and carrier diagnosis. Aim:,To develop a rapid and effective system for carrier detection and prenatal diagnosis of hemophilia A based on a single-multiplexed polymerase chain reaction (PCR) reaction utilizing five microsatellite markers. Patients and methods:,Two intronic microsatellites and three other markers flanking the factor VIII gene were ascertained, and primers were designed for multiplex PCR amplification. A kindred with Hemophilia A was tested for linkage using the panel of primers, and informativity in the general population was ascertained by testing 50 unrelated females. Results:,Co-amplification of all microsatellites was optimized using DNA extracted by standard methods. Rapid detection and sizing of products were carried out using an automated DNA sequencer. The combined microsatellite panel was informative in each of the kindreds tested, and in 100% of the 50 unrelated females (95% CI 94.2,100%). Conclusions:,This method enables the indirect detection of hemophilia A for patients in whom mutations cannot be found, facilitating carrier testing and prenatal analysis. It is rapid and straightforward compared with many other published protocols, and offers a high degree of informativity. [source] Rapid detection of Haemophilus influenzae by hel gene polymerase chain reactionLETTERS IN APPLIED MICROBIOLOGY, Issue 3 2003M.C. Yadav Abstract Aims: To evaluate the efficiency of hel gene polymerase chain reaction (PCR) to detect Haemophilus influenzae in various clinical/non-clinical samples. Methods and Results: Seventy-four clinical samples (cerebrospinal fluid, blood, sputum, throat and nasal swabs) and throat swabs of 17 asymptomatic carriers were collected. Primers were used to amplify the hel gene of H. influenzae encoding P4 outer membrane protein directly from the processed samples. The samples were also examined by conventional culture methods and the results were compared with those of PCR. The culture methods showed positive results in 60 (65·9%) of 91 samples in contrast to 62 (68·12%) samples tested positive by PCR. None of the culture-positive samples were PCR-negative while two of the culture-negative samples were PCR-positive. The specificity of the products was confirmed by Southern hybridization and failure of various other organisms to amplify the hel gene product. The sensitivity of the PCR assay was found to be 50 pg of DNA. Conclusions: These findings suggest that the hel gene PCR is a rapid, sensitive and a specific new method for direct identification of H. influenzae. Significance and Impact of the study: Thus, this PCR test can improve the detection rate of H. influenzae in suspected clinical samples as compared with that of conventional culture methods. [source] Rapid detection of six common Mediterranean and three non-Mediterranean ,-thalassemia point mutations by reverse dot blot analysisAMERICAN JOURNAL OF HEMATOLOGY, Issue 3 2003Enrica Foglietta Abstract We describe the implementation of reverse dot blot (RDB) hybridization as a rapid nonradioactive method for the identification of six frequent globin gene point mutations in the Mediterranean population: ,Hph,: ,2 IVS I donor site GGTGAGG , GG-----; ,NcoI,: ,2 initiation codon ATG , ACG; ,TSaudi,: ,2Poly A signal AATAA , AATAAG; ,Icaria,: ,2 termination codon TAA , AAA (Ter , LYS); ,CS,: ,2 termination codon TAA , CAA (Ter , gly); ,,NcoI: ,1 initiation codon ATG , GTG; and three ,2 globin gene point mutations found in immigrants in Italy: ,T-Quongsze,: ,2 codon 12 CTG , CCG (Leu , Pro); ,Seal Rock,: ,2 termination codon TAA , GAA (TER , GLU); and ,Koyadora,: ,2 termination codon TAA , TCA (TER , SER). The method uses the principle of allele-specific oligonucleotide (ASO) hybridization, but it is a nonradioactive method and permits rapid and simultaneous typing of point mutations and small deletions. Am. J. Hematol. 74:191,195, 2003. © 2003 Wiley-Liss, Inc. [source] Rapid detection and characterization of reactive drug metabolites in vitro using several isotope-labeled trapping agents and ultra-performance liquid chromatography/time-of-flight mass spectrometryRAPID COMMUNICATIONS IN MASS SPECTROMETRY, Issue 6 2009Timo Rousu Reactive metabolites are believed to be one of the main reasons for unexpected drug-induced toxicity issues, by forming covalent adducts with cell proteins or DNA. Due to their high reactivity and short lifespan they are not directly detected by traditional analytical methods, but are most traditionally analyzed by liquid chromatography/tandem mass spectrometry (LC/MS/MS) after chemical trapping with nucleophilic agents such as glutathione. Here, a simple but very efficient assay was built up for screening reactive drug metabolites, utilizing stable isotope labeled glutathione, potassium cyanide and semicarbazide as trapping agents and highly sensitive ultra-performance liquid chromatography/time-of-flight mass spectrometry (UPLC/TOFMS) as an analytical tool. A group of twelve structurally different compounds was used as a test set, and a large number of trapped metabolites were detected for most of them, including many conjugates not reported previously. Glutathione-trapped metabolites were detected for nine of the twelve test compounds, whereas cyanide-trapped metabolites were found for eight and semicarbazide-trapped for three test compounds. The high mass accuracy of TOFMS provided unambiguous identification of change in molecular formula by formation of a reactive metabolite. In addition, use of a mass defect filter was found to be a usable tool when mining the trapped conjugates from the acquired data. The approach was shown to provide superior detection sensitivity in comparison to traditional methods based on neutral loss or precursor ion scanning with a triple quadrupole mass spectrometer, and clearly more efficient detection and characterization of reactive drug metabolites with a simpler test setup. Copyright © 2009 John Wiley & Sons, Ltd. [source] Management of cutaneous tuberculosisDERMATOLOGIC THERAPY, Issue 3 2008Evangeline B Handog ABSTRACT: Cutaneous tuberculosis (TB) is an extrapulmonary form of tuberculosis, which may be classified based on the immunologic state of the host. Chemotherapy still remains the treatment of choice. The management of cutaneous TB follows the same guidelines as that of TB of other organs, which can be treated with a short course four-agent chemotherapeutic regimen given for 2 months followed by a two-drug regimen for the next 4 months. This chapter highlights current treatment recommendations for cutaneous TB. The important factors to consider in the choice of optimal treatment includes the type of cutaneous involvement, stage of the disease, level of immunity, and general condition of the patient. The highest priority in any cutaneous TB control program is the proper, accurate, and rapid detection of cases and the availability of chemotherapy to all tuberculosis patients until cure. Contact tracing is also an important component of efficient tuberculosis control. [source] Label-Free and Ultra-Low Level Detection of Salmonella enterica Serovar Typhimurium Using Electrochemical Impedance SpectroscopyELECTROANALYSIS, Issue 20 2009Jeffrey Abstract An immunosensor for rapid and low level detection of the bacterial pathogen Salmonella enterica Serovar Typhimurium was designed and developed based upon label-free electrochemical impedance spectroscopy and correlated to viable cell counts. The immunosensor was fabricated by electroplating gold onto a disposable printed circuit board (PCB) electrode by immobilizing monoclonal antibody (MAb) specific against Salmonella typhimurium cell surface lipopolysaccharide (LPS) onto the surface of the electrode. Use of mass-fabricated and electroplated PCB electrodes allowed for disposable, highly sensitive, and rapid detection of Salmonella in an aqueous environment. Results demonstrate that in purified solution, Salmonella can be detected as low as 10 CFU in a 100,,L volume and label-free and rapid manner in fewer than 90,s. The cost effective approach described here can be used for detection of pathogens with relevance for healthcare, food, and environmental applications. [source] Direct and Rapid Detection of Diphtherotoxin via Potentiometric Immunosensor Based on Nanoparticles Mixture and Polyvinyl Butyral as MatrixesELECTROANALYSIS, Issue 24 2005Dianping Tang Abstract In this paper a novel potentiometric immunosensor for direct and rapid detection of diphtherotoxin (D-Ag) has been developed by means of self-assembly of monoclonal diphtheria antibody (D-Ab) onto a platinum electrode based on nanoparticles mixture (containing gold nanoparticles and silica nanoparticles) and polyvinyl butyral (PVB) as matrixes. At first, D-Ab was absorbed onto the surface of nanoparticles mixture, and then they were entrapped into polyvinyl butyral sol-gel network on a platinum electrode. The detection is based on the change in the potentiometric response before and after the antigen-antibody reaction in a phosphate buffer solution (pH,7.0). The immobilized D-Ab exhibited direct potentiometric response toward D-Ag. In comparison to the conventional applied methods, this strategy could allow antibodies immobilized with higher loading amount and better retained immunoactivity, as demonstrated by potentiometric response, cyclic voltammetry and electrochemical impedance spectroscopy of the immunosensor. The immunosensor with nanoparticles mixture exhibited much higher sensitivity, better reproducibility, and long-term stability than that with gold nanoparticles or silica nanoparticles alone. The linear range was from 5.0×10,3 to 1.2,,g,mL,1 with a detection limit of 1.1×10,3,,g,mL,1. Up to 16 successive assay cycles with retentive sensitivity were achieved for the probes regenerated with in 0.2,mol,L,1 glycine-hydrochloric acid (Gly-HCl) buffer solution and 0.25,mol,L,1 NaCl. Moreover, the immunosensor with nanoparticles mixture was applied to evaluate a number of practical specimens with potentiometric results in acceptable agreement with those given by the ELISA method, implying a promising alternative approach for detecting diphtherotoxin in the clinical diagnosis. [source] Multiplex primer extension analysis for rapid detection of major European mitochondrial haplogroupsELECTROPHORESIS, Issue 19 2006Martina Wiesbauer Abstract The evolution of the human mitochondrial genome is reflected in the existence of ethnically distinct lineages or haplogroups. Alterations of mitochondrial DNA (mtDNA) have been instrumental in studies of human phylogeny, in population genetics, and in molecular medicine to link pathological mutations to a variety of human diseases of complex etiology. For each of these applications, rapid and cost effective assays for mtDNA haplogrouping are invaluable. Here we describe a hierarchical system for mtDNA haplogrouping that combines multiplex PCR amplifications, multiplex single-base primer extensions, and CE for analyzing ten haplogroup-diagnostic mitochondrial single nucleotide polymorphisms. Using this rapid and cost-effective mtDNA genotyping method, we were able to show that within a large, randomly selected cohort of healthy Austrians (n,=,1172), mtDNAs could be assigned to all nine major European haplogroups. Forty-four percent belonged to haplogroup H, the most frequent haplogroup in European Caucasian populations. The other major haplogroups identified were U (15.4%), J (11.8%), T (8.2%) and K (5.1%). The frequencies of haplogroups in Austria is within the range observed for other European countries. Our method may be suitable for mitochondrial genotyping of samples from large-scale epidemiology studies and for identifying markers of genetic susceptibility. [source] Use of a high-throughput umu -microplate test system for rapid detection of genotoxicity produced by mutagenic carcinogens and airborne particulate matterENVIRONMENTAL AND MOLECULAR MUTAGENESIS, Issue 1 2004Yoshimitsu Oda Abstract In the present study, we developed a rapid umu -microplate test system that uses the nitroreductase- and O -acetyltransferase-overproducing Salmonella typhimurium strain NM3009 and the O -acetyltransferase-overproducing S. typhimurium strain NM2009 to detect genotoxic activity in small volume samples. The assay was used to test the genotoxicity of several standard mutagens and environmental samples. Exponentially growing cultures of NM3009, NM2009, and the parental strain TA1535/pSK1002 were incubated in 96-well microplates with test chemicals both in the presence and in the absence of rat liver S9. The relative ,-galactosidase activities were then determined colorimetrically using either chlorophenol red-,- D -galactopyranoside (CPRG) or O -nitrophenyl-,- D -galactopyranoside (ONPG) as a measure of umuC gene induction activity. The sensitivities of NM3009 without S9 mix and NM2009 with S9 mix to nitroarenes and aromatic amines were up to 24- to 75-fold higher than those of the parent strain. Induction of umuC gene expression was detected more readily with CPRG than ONPG. The umu -microplate assay also detected genotoxicity in organic extracts of particulate matter from air samples collected in Osaka City, Japan. The pattern of the responses suggested that the genotoxic activity of the particulate extract was due primarily to nitrated polycyclic aromatic hydrocarbons. Our results indicate that the umu -microplate assay may be a useful way of carrying out rapid screens for genotoxicity in small-volume environmental samples. Environ. Mol. Mutagen. 43:10,19, 2004. © 2004 Wiley-Liss, Inc. [source] Rapid detection of yeast rRNA genes with primed in situ (PRINS) labelingFEMS YEAST RESEARCH, Issue 4 2009Maciej Wnuk Abstract In yeast, rRNA genes can be detected with the FISH technique using rRNA gene probes. This technique yields reliable, reproducible and precise results, but is time-consuming. Here, the primed in situ DNA synthesis (PRINS) procedure has been optimized for rapid detection of yeast rRNA genes. PRINS, which is as sensitive as PCR and allows cytological localization of analyzed sequences, can be adapted for various screening tests requiring fast labeling of rRNA genes. [source] A novel genotypic test for rapid detection of multidrug-resistant Mycobacterium tuberculosis isolates by a multiplex probe arrayJOURNAL OF APPLIED MICROBIOLOGY, Issue 4 2007S.-L. Zhang Abstract Aims:, To develop and evaluate a novel genotypic test for rapid detection of rifampicin and isoniazid resistance of multidrug-resistant (MDR) Mycobacterium tuberculosis isolates by a multiplex probe array. Methods and Results:, A multiplex probe array was designed for genotypic test to simultaneously screen the mutations of rpoB, katG, inhA and ahpC genes, associated with rifampin and isoniazid resistance in M. tuberculosis, with a probe detecting one of the recently confirmed genetic markers of isoniazid resistance ahpC -6 and -9 locus added. By using the genotypic test developed, 52 MDR isolates were identified, among which 46 isolates had mutations in rpoB (88·5%) and 45 at codon 315 of katG, regulatory region of inhA and oxyR - ahpC intergenic region (86·5%), whereas all 35 susceptible isolates identified showed a wild-type hybridization pattern. The sensitivity and specificity were 88·5% and 100% for rifampicin resistance, and 86·5% and 100% for isoniazid resistance, respectively. Conclusion:, A rapid and simultaneous detection of rifampicin and isoniazid resistance caused by the mutations of rpoB, katG, inhA and ahpC genes in M. tuberculosis isolates could be achieved by a multiplex probe array developed. Significance and Impact of the Study:, This genotypic test protocol has the potential to be developed on clinical application for the rapid detection of drug resistant M. tuberculosis isolates before an efficient chemotherapy is initiated. [source] Evaluation of novel fluorogenic substrates for the detection of glycosidases in Escherichia coli and enterococciJOURNAL OF APPLIED MICROBIOLOGY, Issue 5 2006J.D. Perry Abstract Aims:, Enzyme substrates based on 4-methylumbelliferone are widely used for the detection of Escherichia coli and enterococci in water, by detection of , -glucuronidase and , -glucosidase activity respectively. This study aimed to synthesize and evaluate novel umbelliferone-based substrates with improved sensitivity for these two enzymes. Methods and Results:, A novel , -glucuronide derivative based on 6-chloro-4-methylumbelliferone (CMUG) was synthesized and compared with 4-methylumbelliferyl- , - d -glucuronide (MUG) using 42 strains of E. coli in a modified membrane lauryl sulfate broth. Over 7 h of incubation, the fluorescence generated from the hydrolysis of CMUG by E. coli was over twice that from MUG, and all of the 38 glucuronidase-positive strains generated a higher fluorescence with CMUG compared with MUG. Neither substrate caused inhibition of bacterial growth in any of the tested strains. Four , -glucosidase substrates were also synthesized and evaluated in comparison with 4-methylumbelliferyl- , - d -glucoside (MU-GLU) using 42 strains of enterococci in glucose azide broth. The four substrates comprised , -glucoside derivatives of umbelliferone-3-carboxylic acid and its methyl, ethyl and benzyl esters. Glucosides of the methyl, ethyl and benzyl esters of umbelliferone-3-carboxylic acid, were found to be superior to MU-GLU for the detection of enterococci, especially after 18 h of incubation, while umbelliferone-3-carboxylic acid- , - d -glucoside was inferior. However, the variability in detectable , -glucosidase activity among the different strains of enterococci in short-term assays using the three carboxylate esters (7 h incubation) may compromise their use for rapid detection and enumeration of these faecal indicator bacteria. Conclusions:, The , -glucuronidase substrate CMUG appears to be a more promising detection system than the various , -glucosidase substrates tested. Significance and Impact of the Study:, The novel substrate CMUG showed enhanced sensitivity for the detection of , -glucuronidase-producing bacteria such as E. coli, with a clear potential for application in rapid assays for the detection of this indicator organism in natural water and other environmental samples. [source] Detection of toxigenic Vibrio cholerae from environmental water samples by an enrichment broth cultivation,pit-stop semi-nested PCR procedureJOURNAL OF APPLIED MICROBIOLOGY, Issue 3 2000J. Theron A pit-stop semi-nested PCR assay for the detection of toxigenic Vibrio cholerae in environmental water samples was developed and its performance evaluated. The PCR technique amplifies sequences within the cholera toxin operon specific for toxigenic V. cholerae. The PCR procedure coupled with an enrichment culture detected as few as four V. cholerae organisms in pure culture. Treated sewage, surface, ground and drinking water samples were seeded with V. cholerae and following enrichment, a detection limit of as few as 1 V. cholerae cfu ml,1 was obtained with amplification reactions from crude bacterial lysates. The proposed method, which includes a combination of enrichment, rapid sample preparation and a pit-stop semi-nested PCR, could be applicable in the rapid detection of toxigenic V. cholerae in environmental water samples. [source] Steroidogenic gene expression in H295R cells and the human adrenal gland: adrenotoxic effects of lindane in vitroJOURNAL OF APPLIED TOXICOLOGY, Issue 6 2006Agneta Oskarsson Abstract The focus on the refinement, reduction and replacement of animal use in toxicity testing requires the development of cell-based systems that mimic the effects of xenobiotics in human tissues. The human adrenocortical carcinoma cell line, H295R, has been proposed as a model for studies on adrenal steroidogenesis and its disruption. In this study, expression profiles for nine adrenal steroidogenic genes were characterized in H295R cells using real-time RT-PCR. Treatment with forskolin increased cortisol secretion and stimulated transcription of all the steroidogenic genes except SULT2A1. The transcript profile from H295R cells in the presence and absence of forskolin was compared with the transcript profile from human adrenal glands. The gene expression pattern observed in the forskolin-treated H295R cells was more similar to that in the human adrenal gland, than the expression pattern in untreated cells. To examine H295R cells as a possible in vitro system for the assessment of adrenal disruption using molecular endpoints, the insecticide lindane (, -hexachlorocyclohexane) was used. In vivo, lindane has been shown to inhibit testicular, ovarian and adrenal steroidogenesis. It was demonstrated that lindane reduced cortisol secretion, downregulated the expression of a subset of the genes encoding steroidogenic enzymes and repressed transcriptional activation of the steroidogenic acute regulatory protein (StAR) gene promoter. Thus the H295R cell line provides a good in vitro system for the analysis of the human adrenal steroidogenic pathway at the level of hormone production and gene expression. This in vitro test can be used for the rapid detection of adrenal endocrine disruption and as a tool for mechanistic studies. Copyright © 2006 John Wiley & Sons, Ltd. [source] Hyperspectral imaging combined with principal component analysis for bruise damage detection on white mushrooms (Agaricus bisporus)JOURNAL OF CHEMOMETRICS, Issue 3-4 2008A. A. Gowen Abstract Hyperspectral imaging (HSI) combines conventional imaging and spectroscopy to simultaneously acquire both spatial and spectral information from an object. This technology has recently emerged as a powerful process analytical tool for rapid, non-contact and non-destructive food analysis. In this study, the potential application of HSI for damage detection on the caps of white mushrooms (Agaricus bisporus) was investigated. Mushrooms were damaged by controlled vibration to simulate damage caused by transportation. Hyperspectral images were obtained using a pushbroom line-scanning HSI instrument, operating in the wavelength range of 400,1000,nm with spectroscopic resolution of 5,nm. The effective resolution of the CCD detector was 580,×,580,pixels by 12 bits. Two data reduction methods were investigated: in the first, principal component analysis (PCA) was applied to the hypercube of each sample, and the second PC (PC 2) scores image was used for identification of bruise-damaged regions on the mushroom surface; in the second method PCA was applied to a dataset comprising of average spectra from regions normal and bruise-damaged tissue. In this case it was observed that normal and bruised tissue were separable along the resultant first principal component (PC 1) axis. Multiplying the PC 1 eigenvector by the hypercube data allowed reduction of the hypercube to a 2-D image, which showed maximal contrast between normal and bruise-damaged tissue. The second method performed better than the first when applied to a set of independent mushroom samples. The results from this study could be used for the development of a non-destructive monitoring system for rapid detection of damaged mushrooms on the processing line. Copyright © 2008 John Wiley & Sons, Ltd. [source] Comparison of ATP production in whole blood and lymphocyte proliferation in response to phytohemagglutininJOURNAL OF CLINICAL LABORATORY ANALYSIS, Issue 5 2007Nancy H. Augustine Abstract Lymphocyte proliferation in response to mitogens, phytohemagglutinin (PHA), concanavalin A, pokeweed, and/or specific antigens has been the method of choice for in vitro diagnosis of cell-mediated immune dysfunction. Recently, an assay to measure intracellular adenosine triphosphate (ATP) production in response to PHA has been developed that requires a shorter, overnight incubation. We compared a standard 5- to 7-day lymphocyte mitogen stimulation assay utilizing tritiated thymidine (3H-thy) incorporation to one in which ATP production in response to PHA by CD4-positive cells is measured in a luminometer that requires only 18,24,hr. A total of 20 patient samples suspected of having decreased cell-mediated immunity submitted for mitogen induced lymphocyte proliferation and 21 normal controls were tested in both assays. A comparison of these two methods has demonstrated that the screening ATP assay has a sensitivity at 24,hr of 100% in detecting decreased PHA induced lymphocyte proliferation at 5 days and a specificity of 85% in the samples obtained from normal controls. The data indicate that the ATP assay may be a useful screening tool for more rapid detection of blood samples with decreased cell-mediated immune responses. However, a positive screen should always be confirmed by 3H-thy uptake using mitogens and recall antigens like candida and tetanus. J. Clin. Lab. Anal. 21:265,270, 2007. © 2007 Wiley-Liss, Inc. [source] Comparison of cell culture with RT-PCR for enterovirus detection in stool specimens from patients with acute flaccid paralysisJOURNAL OF CLINICAL LABORATORY ANALYSIS, Issue 4 2007Zabih-Ollah Shoja Abstract Since October 2000, Iran has been declared polio-free by the World Health Organization (WHO). Despite the fact that poliomyelitis caused by polioviruses has been eliminated from Iran, the number of acute flaccid paralysis (AFP) cases has not been reduced. Therefore, it is of great importance to investigate the other viral agents that may cause AFP (mainly nonpolio enteroviruses, which play a significant role in the etiology of neurological syndromes). Some enteroviruses do not grow in the conventional cell lines that are being used for enterovirus detection. Furthermore, the virus titer is an important factor in the sensitivity of cell culture to detect the virus. The fact that cell culture is a time-consuming procedure is another reason to find a more practical method for enterovirus detection. Therefore, a more sensitive and rapid method should be used to detect enteroviruses as efficiently as possible in the stool specimens of AFP cases. The aim of this study was to evaluate cell culture and RT-PCR in enterovirus detection. Findings have shown that RT-PCR can increase the rate of nonpolio enterovirus detection by up to 10% in comparison with cell culture. Also, the rapid detection of enteroviruses by RT-PCR can decrease both the unnecessary use of antibiotics and the costs in clinical practice. For this reason, we find that RT-PCR is a more practical technique for enterovirus detection. J. Clin. Lab. Anal. 21:232,236, 2007. © 2007 Wiley-Liss, Inc. [source] Development of a single-tube PCR-pyrosequencing method for the simultaneous and rapid detection of four variant alleles of CYP2C9 gene polymorphismJOURNAL OF CLINICAL PHARMACY & THERAPEUTICS, Issue 2 2008Y. Okada MS Summary Background and Objective:, CYP2C9 is a polymorphic enzyme that has been reported to metabolize several clinically useful drugs such as warfarin, phenytoin and non-steroidal anti-inflammatory drugs. We designed a rapid single-tube multiplex assay to detect four variant alleles of the CYP2C9 in a single polymerase chain reaction (PCR) and a single pyrosequencing reaction. Methods:, A multiplex PCR was designed to amplify two fragments simultaneously, one containing 430C>T (CYP2C9*2) polymorphism and other containing 1075A>C (CYP2C9*3), 1076T>C (CYP2C9*4) and 1080C>G (CYP2C9*5) polymorphisms. Results:, Four variants of the CYP2C9 gene could be simultaneously detected using only two varieties of pyrosequencing primers in a single-tube. The success rate for the four SNPs (*2, *3,*4 and *5) was high. Genotypes obtained by the multiplex reaction were 100% concordant with genotypes obtained using direct DNA sequencing (n = 96). The analysis time was halved, compared with existing simplex pyrosequencing. The system allowed high-throughput analysis of over 384 samples per hour. Discussion:, Our method reduces running cost and halves analysis time, compared to simplex pyrosequencing. Another advantage of this method is that it analyses and determines multiple bases around the polymorphic site thereby reducing the possibility of scoring a truncated PCR product. [source] | |||||||||||||||||||||||||||||||