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1.
Forensic Sci Int ; 275: 160-166, 2017 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-28366623

RESUMO

Food poisoning is frequently caused by the accidental ingestion of toxic plants that possess strong morphological similarities to edible plants. False helleborine (Veratrum album) is one of the most common plants involved in such accidents. In cases of poisoning by toxic plants, rapid and accurate identification, usually based on the morphological or chemical analysis of plant parts, is required for appropriate medical treatment or forensic investigation. However, morphological examinations require experience in systematic botany because the samples are fragmentary, and chemical analysis of natural compounds can be difficult. In this study, we developed a TaqMan real-time PCR method using trnH-psbA and trnL-trnF that could be carried out in 30-60min. The lower detection limit was less than 10pg of DNA and the primer sets were specific to V. album and Veratrum stamineum. Mixed samples, cooked samples, and simulated gastric contents were successfully identified, and a multiplex assay of two regions was also possible. These results indicate that the TaqMan real-time PCR analysis is a very effective method to detect small samples of V. album and V. stamineum accurately and rapidly in poisoning cases.


Assuntos
Plantas Tóxicas/genética , Reação em Cadeia da Polimerase em Tempo Real/métodos , Veratrum/genética , DNA de Plantas/genética , Doenças Transmitidas por Alimentos , Humanos , Reação em Cadeia da Polimerase Multiplex/métodos
2.
Mol Biotechnol ; 58(3): 212-9, 2016 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-26800992

RESUMO

Species identification from extracted DNA is sometimes needed for botanical samples. DNA quantification is required for an accurate and effective examination. If a quantitative assay provides unreliable estimates, a higher quantity of DNA than the estimated amount may be used in additional analyses to avoid failure to analyze samples from which extracting DNA is difficult. Compared with conventional methods, real-time quantitative PCR (qPCR) requires a low amount of DNA and enables quantification of dilute DNA solutions accurately. The aim of this study was to develop a qPCR assay for quantification of chloroplast DNA from taxonomically diverse plant species. An absolute quantification method was developed using primers targeting the ribulose-1,5-bisphosphate carboxylase/oxygenase large subunit (rbcL) gene using SYBR Green I-based qPCR. The calibration curve was generated using the PCR amplicon as the template. DNA extracts from representatives of 13 plant families common in Japan. This demonstrates that qPCR analysis is an effective method for quantification of DNA from plant samples. The results of qPCR assist in the decision-making will determine the success or failure of DNA analysis, indicating the possibility of optimization of the procedure for downstream reactions.


Assuntos
DNA de Cloroplastos/análise , Reação em Cadeia da Polimerase em Tempo Real/métodos , Código de Barras de DNA Taxonômico/métodos , Primers do DNA/genética , DNA de Cloroplastos/química , DNA de Cloroplastos/classificação , Compostos Orgânicos/química , Plantas/classificação , Plantas/genética
4.
Anal Chem ; 86(9): 4316-26, 2014 May 06.
Artigo em Inglês | MEDLINE | ID: mdl-24678766

RESUMO

A highly sensitive and specific real-time field-deployable detection technology, based on counterflow air introduction atmospheric pressure chemical ionization, has been developed for a wide range of chemical warfare agents (CWAs) comprising gaseous (two blood agents, three choking agents), volatile (six nerve gases and one precursor agent, five blister agents), and nonvolatile (three lachrymators, three vomiting agents) agents in air. The approach can afford effective chemical ionization, in both positive and negative ion modes, for ion trap multiple-stage mass spectrometry (MS(n)). The volatile and nonvolatile CWAs tested provided characteristic ions, which were fragmented into MS(3) product ions in positive and negative ion modes. Portions of the fragment ions were assigned by laboratory hybrid mass spectrometry (MS) composed of linear ion trap and high-resolution mass spectrometers. Gaseous agents were detected by MS or MS(2) in negative ion mode. The limits of detection for a 1 s measurement were typically at or below the microgram per cubic meter level except for chloropicrin (submilligram per cubic meter). Matrix effects by gasoline vapor resulted in minimal false-positive signals for all the CWAs and some signal suppression in the case of mustard gas. The moisture level did influence the measurement of the CWAs.


Assuntos
Poluentes Atmosféricos/análise , Substâncias para a Guerra Química/análise , Espectrometria de Massas em Tandem/métodos , Pressão Atmosférica , Limite de Detecção
5.
Biosens Bioelectron ; 24(11): 3299-305, 2009 Jul 15.
Artigo em Inglês | MEDLINE | ID: mdl-19450964

RESUMO

A semi-automated bacterial spore detection system (BSDS) was developed to detect biological threat agents (e.g., Bacillus anthracis) on-site. The system comprised an aerosol sampler, micro-fluidic chip-A (for spore germination and cell lysis), micro-fluidic chip-B (for extraction and detection of genomic DNA) and an analyzer. An aerosol with bacterial spores was first collected in the collection chamber of chip-A with a velocity of 300 l/min, and the chip-A was taken off from the aerosol sampler and loaded into the analyzer. Reagents packaged in the chip-A were sequentially applied into the chamber. The genomic DNA extract from spore lyzate was manually transferred from chip-A to chip-B and loaded into the analyzer. Genomic DNA in chip-B was first trapped on a glass bead column, washed with various reagents, and eluted to the detection chamber by sequential auto-dispensing. Isothermal and chimeric primer-initiated amplification of nucleic acids (ICAN) with fluorescent measurement was adopted to amplify and detect target DNA. Bacillus subtilis was the stimulant of biological warfare agent in this experiment. Pretreatment conditions were optimized by examining bacterial target DNA recovery in the respective steps (aerosol collection, spore germination, cell lysis, and DNA extraction), by an off-chip experiment using a real-time polymerase chain reaction quantification method. Without the germination step, B. subtilis spores did not demonstrate amplification of target DNA. The detection of 10(4) spores was achieved within 2h throughout the micro-fluidic process.


Assuntos
Aerossóis/análise , Bacillus anthracis/genética , Bacillus anthracis/isolamento & purificação , Técnicas Biossensoriais/instrumentação , DNA Bacteriano/análise , Técnicas Analíticas Microfluídicas/instrumentação , Técnicas de Amplificação de Ácido Nucleico/instrumentação , Microbiologia do Ar , Algoritmos , DNA Bacteriano/genética , Monitoramento Ambiental/instrumentação , Monitoramento Ambiental/métodos , Desenho de Equipamento , Análise de Falha de Equipamento , Técnicas de Amplificação de Ácido Nucleico/métodos , Esporos Bacterianos/genética , Esporos Bacterianos/isolamento & purificação
6.
Artigo em Inglês | MEDLINE | ID: mdl-16569519

RESUMO

To verify the exposure to nerve gas, a method for detecting human butyrylcholinesterase (BuChE)-nerve gas adduct was developed using LC-electrospray mass spectrometry (ESI-MS). Purified human serum BuChE was incubated with sarin, soman or VX, and the adduct was purified by sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) and digested in gel by treatment with chymotrypsin. The resulting peptide mixture was subjected to LC-ESI-MS. From the chymotryptic digest of untreated human BuChE, one peak corresponding to the peptide fragment containing the active center serine residue was detected on the extracted ion chromatogram at m/z 948.5, and the sequence was ascertained to be "GESAGAASVSL" by MS/MS analysis. From the chymotryptic digest of the human BuChE-sarin adduct, a singly charged peptide peak was detected on the extracted ion chromatogram at m/z 1,069.5, and the sequence was ascertained to be "GEXAGAASVSL" by MS/MS analysis (X denotes isopropylmethylphosphonylated serine). The difference in molecular weight (120.0 Da) between the active center peptide fragments corresponding to the untreated BuChE and BuChE-sarin adduct was assumed to be derived from the addition of an isopropyl methylphosphonyl moiety to the serine residue. The formation of human BuChE adducts with soman, VX and an aged soman adduct was confirmed by detecting the respective active center peptide fragments using LC-ESI-MS. To apply the established method to an actual biological sample, human serum was incubated with VX, and the adduct was purified by procainamide affinity chromatography followed by SDS-PAGE. After chymotryptic in gel digestion, the ethylphosphonylated active center peptide fragment could be detected, and the structure of the residue was ascertained by LC-ESI-MS analysis.


Assuntos
Butirilcolinesterase/sangue , Substâncias para a Guerra Química/química , Cromatografia Líquida de Alta Pressão/métodos , Quimotripsina/metabolismo , Espectrometria de Massas por Ionização por Electrospray/métodos , Sequência de Aminoácidos , Butirilcolinesterase/química , Butirilcolinesterase/metabolismo , Inibidores da Colinesterase/química , Eletroforese em Gel de Poliacrilamida/métodos , Humanos , Dados de Sequência Molecular , Estrutura Molecular , Compostos Organotiofosforados/química , Sarina/química , Soman/química
7.
J Chromatogr A ; 1061(2): 235-41, 2004 Dec 24.
Artigo em Inglês | MEDLINE | ID: mdl-15641367

RESUMO

A method for determining thiodiglycol (TDG), a mustard gas hydrolysis product in water, serum and urine samples using gas chromatography-mass spectrometry (GC-MS) after tert-butyldimethylsilylation (TBDMS) is described. Quantitation of TDG was performed by measuring the respective peak area on the extracted ion chromatogram of m/z 293, using an internal standard, the TDG homologue, thiodipropanol, peak area of which was measured as m/z 321. The presence of salts in the sample solution not only suppressed the loss of TDG by vaporization during the evaporation of water, but also facilitated the rate of production of di-silylated derivative, bis(tert-butyldimethylsilyoxylethyl)sulfide (TDG-(TBDMS)2). Under the pretreatment conditions used, in which 0.5 ml of water sample supplemented with 100 microM potassium chloride was evaporated to dryness under reduced pressure, followed by reaction with N-methyl-N-(tert-butyldimethylsilyl)trifluoroacetamide at 60 degrees C for 1 h, TDG-(TBDMS)2 was reproducibly detected with about a 55% recovery and a limit of detection (LOD, scan mode, S/N = 3) of 5.4 ng/ml. TDG was also determined by GC-MS from a 0.5 ml serum sample (after perchloric acid deproteinization) and from a 0.1 ml urine sample, after TBDMS derivatization. The LOD was determined to be 7.0 and 110 ng/ml for serum and urine, respectively.


Assuntos
Cromatografia Gasosa-Espectrometria de Massas/métodos , Gás de Mostarda/química , Silanos/química , Compostos de Sulfidrila/análise , Hidrólise , Sensibilidade e Especificidade , Compostos de Sulfidrila/sangue , Compostos de Sulfidrila/urina
8.
J Agric Food Chem ; 50(16): 4445-51, 2002 Jul 31.
Artigo em Inglês | MEDLINE | ID: mdl-12137458

RESUMO

A gas chromatographic-mass spectrometric method for the determination of S-methyl-L-cysteine sulfoxide (1), S-propyl-L-cysteine sulfoxide (2), and S-propenyl-L-cysteine sulfoxide (3), specific marker compounds in the genus Allium, is described. The target amino acids were converted to the tert-butyldimethylsilyl derivatives. The products were silylated on the amino and carboxyl groups and on an additional oxygen atom and were separated on a nonpolar capillary column. That incorporation of three tert-butyldimethylsilyl groups had occurred was verified by mass spectrometry, which gave an m/z 302 fragment as base peak (amino acid side chain eliminated ion) and m/z 436 (1), 464 (2), or 462 (3) as major peaks (tert-butyl function eliminated ion), by electron impact ionization. The detection limits for 1 and 2 under selected ion monitoring at m/z 436 (1) and m/z 464 (2), respectively, were determined to be 0.3 and 1.8 ng per injection. To clean up the analytes from the solvent extract of onion, as a representative food material, onion, the sample solution was subjected to combined solid phase extraction. The eluate from a Sep-Pak C(18) cartridge was applied to a Bond Elut SCX cartridge (H(+) form), followed by washing with 0.1 M hydrochloric acid and elution with 0.5 M ammonia. From a simulated matrix solution containing 5% sucrose, 1 and 2 were extracted quantitatively, and the detection yield was approximately 75%. The contents of 1, 2, and 3 in commercial onion were estimated to be 0.3, 3.1, and 3.0 mg, respectively, per gram of fresh weight.


Assuntos
Cisteína/análogos & derivados , Cisteína/análise , Cromatografia Gasosa-Espectrometria de Massas/métodos , Cebolas/química , Silanos/química , Sulfóxidos/análise , Cromatografia Líquida de Alta Pressão , Cisteína/química , Sulfóxidos/química
9.
Artigo em Inglês | MEDLINE | ID: mdl-12127328

RESUMO

In order to confirm that diisopropylfluorophosphate (DFP) phosphorylates the active site serine residue in alpha-chymotrypsin, a peptide containing the phosphorylated active site was analyzed by liquid chromatography (LC)-electrospray mass spectrometry (ESI-MS). After reduction with dithiothreitol and subsequent alkylation with acrylamide, alpha-chymotrypsin was digested by treatment with trypsin. Tryptic digest was subjected to LC-ESI-MS. Nearly all the peptide fragments were identified by comparison with fragments predicted from as tryptic digest of alpha-chymotrypsin. From the tryptic digest of native alpha-chymotrypsin, a doubly protonated peptide peak which corresponded to the peptide fragment containing the active site serine residue was detected on a selected ion chromatogram at m/z 1265.0, and the sequence was determined to be "DAMICAGASGVSSCMGDSGGPLVCK". From the tryptic digest of DFP-inhibited alpha-chymotrypsin, the doubly protonated peptide peak was detected on a selected ion chromatogram at m/z 1347.0. The difference in mass number (82 in a doubly charged ion) of active site peptide fragments between the native and DFP inhibited alpha-chymotrypsins was assumed to be the result of phosphorylation of the serine residue with a diisopropylphosphoryl moiety. A total of +164 Da mass shifts of y-series fragment ions from the y(8) to y(21) positions in the active site peptide of the DFP inhibited alpha-chymotrypsin was observed, in comparison with the native alpha-chymotrypsin. Thus, the phosphorylation site in alpha-chymotrypsin could be unequivocally identified to be at the serine residue which is located at position 47, from the N-terminus of the alpha-chymotrypsin C-chain.


Assuntos
Cromatografia Líquida/métodos , Compostos Organofosforados/análise , Fragmentos de Peptídeos/química , Serina Endopeptidases/química , Espectrometria de Massas por Ionização por Electrospray/métodos , Sequência de Aminoácidos , Dados de Sequência Molecular
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