Asunto(s)
Brotes de Enfermedades , Infecciones por Enterobacteriaceae/epidemiología , Escherichia/clasificación , Escherichia/aislamiento & purificación , Enfermedades Transmitidas por los Alimentos/epidemiología , Técnicas de Tipificación Bacteriana , ADN Bacteriano/genética , Electroforesis en Gel de Agar , Infecciones por Enterobacteriaceae/microbiología , Escherichia/genética , Escherichia/fisiología , Enfermedades Transmitidas por los Alimentos/microbiología , Humanos , Reacción en Cadena de la PolimerasaRESUMEN
A rapid multiplex real-time PCR assay was developed to achieve highly specific, simultaneous detection of two kinds of mushrooms, Omphalotus guepiniformis and Lentinula edodes. Primers and TaqMan minor groove binder probes were designed according to the internal transcribed spacers 1-5.8S region of rDNA and evaluated by the specificity for fruiting bodies of 17 O. guepiniformis, 16 L. edodes and samples from 57 other species. DNA extracts of all the target species had positive signals with no cross-reaction, the limit of detection being 0.00025 ng of DNA. Threshold cycle (Ct) values for raw and processed fruiting bodies and for fruiting bodies (1% (w/w)) mixed with foodstuffs or artificial gastric juice contents ranged from 17.16 to 26.60 for both examined species. This new assay proved specific to the target species, highly sensitive, and applicable to processed food samples and gastric juice contents, making it useful for rapidly identifying O. guepiniformis and L. edodes.
Asunto(s)
Agaricales/aislamiento & purificación , ADN de Hongos/aislamiento & purificación , Cuerpos Fructíferos de los Hongos/aislamiento & purificación , Reacción en Cadena de la Polimerasa Multiplex/métodos , Reacción en Cadena en Tiempo Real de la Polimerasa/métodos , Hongos Shiitake/aislamiento & purificación , Agaricales/genética , Cartilla de ADN/genética , Sondas de ADN/genética , ADN de Hongos/genética , ADN Espaciador Ribosómico/genética , ADN Espaciador Ribosómico/aislamiento & purificación , Análisis de los Alimentos , Cuerpos Fructíferos de los Hongos/genética , Jugo Gástrico/química , Sensibilidad y Especificidad , Hongos Shiitake/genéticaRESUMEN
A rapid and sensitive TaqMan real-time PCR assay for the detection of Pacific cod (Gadus macrocephalus) and capelin (Mallotus villosus) roes in Alaska pollack (Theragra chalcogramma) roe product was developed. The primers and the TaqMan MGB (minor groove binder) probes were designed based on the gene encoding cytochrome b for the specific detection of Alaska pollack, Pacific cod and capelin. This real-time PCR assay had the detection limit of 0.002 ng/microL mitochondrial DNA and showed no cross-reaction with 48 other species. The calculated r(2) values of the standard curves for the three species were 1.000. This assay was applied for the detection of Pacific cod and capelin roes in mixture samples: Pacific cod or capelin roes were added to Alaska pollack roes at 0.1, 1 and 10%. The threshold cycle values were obtained from both of the mixture samples at 0.1%. Practical applicability of this assay was examined with 64 samples of Alaska pollack roe products. In all cases, the species detected from the samples corresponded with species described on the food label.
Asunto(s)
Productos Pesqueros , Peces , Gadiformes , Alaska , Animales , Sistemas de Computación , Citocromos b/genética , ADN Mitocondrial/análisis , Peces/genética , Gadiformes/genética , Reacción en Cadena de la PolimerasaRESUMEN
A method for the determination of nonvolatile amines (putrescine, cadaverine, histamine, tyramine and spermidine) in foods by solid-phase extraction and excimer-forming derivatization was investigated. Nonvolatile amines in a solid sample were extracted with 3% trichloroacetic acid, and the amines in a liquid sample were extracted with water. The extract was applied to polymer-based strong cation exchange mini-column, which was then rinsed with phosphate buffer of pH 6.8 and water. Nonvolatile amines were eluted with 100 mmol/L potassium carbonate solution. The solution was mixed with 6 mmol/L 1-pyrenebutylyl chloride solution and derivatized. Derivatives of nonvolatile amines were analyzed by LC-FLD, and the identity of the amines was confirmed by LC-MS/MS without derivatization. The limit of detection (S/N> or =3) of nonvolatile amines in all samples was 0.04 microg/g, and the limit of quantitation (S/N> or =10) was 0.1 microg/g. Recoveries of nonvolatile amines from fish tissues, miso, shoyu and red wine were in the range of 80.4-111%.
Asunto(s)
Aminas/análisis , Cromatografía Liquida , Análisis de los Alimentos/métodos , Extracción en Fase Sólida , Cadaverina/análisis , Fluorescencia , Histamina/análisis , Putrescina/análisis , Espermidina/análisis , Triptaminas/análisisRESUMEN
Reverse transcription-PCR targeting the VP0 gene of human parechoviruses (HPeVs) was used to identify two isolates from two Japanese children's stool specimens. Molecular analysis revealed that these isolates belonged to HPeV type 4, and their nucleotide identity in the P1 region was 85.0%.
