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Sample records for detectado por pcr

  1. Características sociodemográficas de mujeres peruanas con virus papiloma humano detectado por PCR-RFLP

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    Yasser Sullcahuaman-Allende

    Full Text Available Con el objetivo de determinar las características sociodemográficas del virus de pacientes con papiloma humano (VPH referidas al Instituto Nacional de Enfermedades Neoplásicas (INEN durante los años 2012-2014, se realizó la detección del VPH en células cervicales por reacción en cadena de la polimerasa (PCR. En 465 muestras cervicales se detectaron 151 (32,5% casos de VPH positivas. Los genotipos más frecuentes fueron VPH-16 (23,8% y VPH-6 (11,9%. La presencia de VPH fue mayor en mujeres de 17 a 29 años (OR 2,64, IC 95%:1,14-6,13 y solteras (OR 2,31, IC 95%: 1,37-3,91, la presencia de genotipos de VPH de alto riesgo fue mayor en solteras (OR 2,19, IC 95%: 1,04-4,62. En conclusión, mujeres jóvenes y solteras presentaron mayor frecuencia de casos VPH-positivos a quienes se debe enfatizar la participación en programas de tamizaje con métodos moleculares y citológicos combinados, a fin de detectar oportunamente el riesgo de desarrollar cáncer de cuello uterino

  2. Oncogenes E6-E7 de los Papilomavirus Humanos de alto riesgo detectados por PCR en Biopsias de pene incluidas en parafina

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    I Guerrero

    1999-01-01

    Full Text Available La alta prevalencia del papilomavirus humano (PVH, referida a nivel mundial, en lesiones genitales de ambos sexos, el rol del varón como reservorio pasivo del virus, y el incremento de la mortalidad por cáncer genital en la mujer en nuestro país, motiva la detección y correlación de los oncogenes de los PVH de alto riesgo con la neoplasia de pene. Informamos de diez casos de biopsias de carcinoma escamoso de pene, incluidos en parafina, los cuales fueron investigados para la presencia de los oncogenes E6-E7 de PVH de alto riesgo, utilizando cebadores tipo específico para los PVH -16 y 18, mediante la reacción en cadena de la polimerasa (PCR. El 40% de los casos mostró un producto de amplificación ADN E6-E7 de los PVH estudiados, correspondiendo el 75% de ellos a detección simple por PVH-18 y el 25% presentó detección mixta ADN E6 - E7 del PVH-16 y 18 simultáneamente. El producto de amplificación fue sometido a comprobación por análisis de restricción específico. La prevalencia obtenida de los oncogenes E6-E7 de los PVH de alto riesgo, usando un método tan sensible como la PCR, apoya el rol de estos virus en el proceso de carcinogénesis de la neoplasia de pene.

  3. Diagnostico por PCR del complejo Sigatoka en Colombia.

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    Romero Magally

    1999-06-01

    Full Text Available Las Sigatokas negra y amarilla son enfermedades causadas por Mycosphaerella fijiensis y Mycosphaerella musicola respectivamente. Estas dos especies de hongos estrechamente relacionadas y morfológicamente similares, causan necrosis severa en las hojas de banano y plátano disminuyendo el área de tejido fotosintético y acelerando la maduración del fruto. Las enfermedades producidas por estos hongos afectan la mayoría de las áreas cultivadas de banano y plátano a nivel mundial, generando un gran problema económico y ambiental. Los síntomas que produce cada uno de estos patógenos en el cultivo son similares, por lo que su correcta identificación en campo se hace difícil. Con el fin de hacer una identificación precisa de cada uno de ellos decidimos estandarizar una prueba diagnóstica basada en la reacción en cadena de la polimerasa (PCR utilizando dos oligonucleótidos de 21 bases (MF 137 y MM 137 específicos para cada una de las especies M. fijiensis y M. musicola respectivamente. MF 137 y MM 137 codifican regiones variables identificadas en las secuencias interespaciadoras (ITS del DNA ribosomal.

  4. Genotipificación de aislamientos de Bartonella bacilliformis por amplificación de elementos repetitivos mediante el uso de REP-PCR y ERIC-PCR

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    Carlos Padilla R

    2003-07-01

    Full Text Available Objetivos: Genotipificar los aislamientos de Bartonella bacilliformis a través de la amplificación de elementos repetitivos mediante el uso de ERIC-PCR y REP-PCR, y determinar si existe variabilidad genética entre aislamientos de varias zonas endémicas. Materiales y Métodos: Se evaluaron mediante el uso del ERIC-PCR y REP-PCR 17 aislamientos de B. bacilliformis de Lima, Cusco y Ancash. Los aislamientos fueron realizados durante los años 1998 y 1999. Para el análisis de los patrones de bandas se usó el software GelCompar 4,0. Resultados: Fueron identificados en los 17 aislamientos 10 genotipos. Los genotipos D, E y H fueron detectados en Cusco; mientras que los genotipos B, C, G, J e I en Lima; y el genotipo F en Ancash. Conclusiones: Nuestros resultados sugieren que REP-PCR y ERIC-PCR son métodos útiles para genotipificar aislamientos de B. bacilliformis. La variabilidad genética debe ser tomada en cuenta en estudios epidemiológicos y clínicos de Bartonelosis; así como el desarrollo de nuevas técnicas diagnósticas y de vacunas.

  5. DETECCION DE Brucella abortus POR PCR EN MUESTRAS DE SANGRE Y LECHE DE VACUNOS

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    Xiomara Mosquera C

    2008-12-01

    Full Text Available Objetivo. Evaluar el uso de la Reacción en Cadena de la Polimerasa (PCR para la detección de Brucella abortus en muestras de sangre y leche de vacunos. Materiales y métodos. Este estudio de tipo descriptivo fue realizado durante los años 2004 y 2005. Se analizaron 136 animales de tres fincas localizadas en el municipio de Durania, Norte de Santander, Colombia. Se evaluó la presencia de anticuerpos en la leche mediante la prueba del anillo (PAL. Se amplificó el fragmento de 223pb del gen BCSP31. Se emplearon los cebadores B4 y B5 de la región interna de la secuencia del gen BCSP31 (GenBank, número M20404. Resultados. En aquellos animales positivos se obtuvo una muestra de sangre y leche para el análisis por PCR, la sangre no fue analizada por serología. Se evaluaron diferentes métodos de extracción de ADN. Se encontró que un 13.2% (18/136 de las muestras de leche fueron positivas a la PAL. Se analizaron 33 muestras de leche negativas por PAL de las cuales el 30.3% (10/33 resultaron positivas por PCR. Al analizar las muestras de sangre de los animales positivos por PAL el 94.1% (16/17 fueron positivas por PCR, mientras que el 47% (8/17 de las muestras de leche positivas por PAL, fueron positivas por PCR. Conclusiones. Se demostró la amplificación de un fragmento de ADN de Brucella abortus en muestras de sangre y leche de vacunos. Los resultados preliminares demostraron que es posible usar PCR como prueba diagnóstica de brucelosis en Colombia.

  6. Survival in soil and detection of co-transformed Trichoderma harzianum by nested PCR Sobrevivência em solo e detecção de co-transformantes de Trichoderma harzianum por PCR "nested"

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    Paulo Roberto Queiroz

    2004-04-01

    Full Text Available The objective of this work was to evaluate the survival of two Trichoderma harzianum co-transformants, TE 10 and TE 41, carrying genes for green fluorescent protein (egfp and for resistance to benomyl, during four weeks in a contained soil microcosm. Selective culture media were used to detect viable fungal material, whose identity was confirmed by the observation of the fluorescent phenotype by direct epifluorence microscopy. PCR using two nested primer pairs specific to the egfp gene was also used to detect the transformed fungi. Although it was not possible to reliably detect the egfp gene directly from soil extracts, an enrichment step involving selective culture of soil samples in liquid medium prior to DNA extraction enabled the consistent detection of the T. harzianum co-transformants by nested PCR for the duration of the incubation period.O objetivo deste trabalho foi avaliar a sobrevivência de dois co-transformantes de Trichoderma harzianum, TE 10 e TE 41, expressando o gene da proteína de fluorescência verde (egfp e resistência a benomil, por um período de quatro semanas em microcosmo de solo, sob condições controladas. Foi utilizado um meio seletivo para detecção de material fúngico viável, o qual foi confirmado por observação quanto ao fenótipo de fluorescência em microscópio de epifluorescência direta. O fungo transformado foi detectado por PCR "nested", utilizando-se dois pares de primers específicos para o gene egfp. Foram utilizados meios líquidos enriquecidos no cultivo de amostras de solo, permitindo uma detecção consistente de co-transformantes de T. harzianum, uma vez que não foi possível a detecção do gene egfp por PCR de amostras de DNA extraídas diretamente de solo.

  7. Diagnóstico de malaria por el método de la PCR anidada

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    Nohora M. Mendoza

    2001-12-01

    Full Text Available En 1998, se presentaron en Colombia 256.697 casos de malaria: 132.712 por Plasmodium falciparum, 121.161 por Plasmodium vivax y 2.824 infecciones mixtas. Dada esta situación, el Programa de Malaria del Laboratorio Nacional de Referencia se propuso evaluar la PCR anidada frente a la gota gruesa, como método alternativo para el diagnóstico de malaria. Se utilizó la reacción en cadena de la polimerasa (PCR anidada para amplificar secuencias variables de genes de la subunidad ribosomal pequeña 18s (ssrARN de P falciparum y P vivax. Para evaluar la PCR, se recolectaron 102 muestras de pacientes febriles en Quibdó (Chocó y se determinó la presencia de parásitos tanto por PCR como por observación microscópica (gota gruesa. Los resultados de las dos pruebas se compararon entre sí. Los criterios de evaluación fueron: precisión, exactitud, limite de detección, sensibilidad, especificidad, valores predictivos positivo (VPP y negativo (VPN e índice kappa. La PCR anidada presentó una sensibilidad general del 100%: 96,7% para Pvivax y 100% para P falciparum. La especificidad general para P vivax y para P falciparum fue del 100%. El VPP general de la prueba fue del 90% y el VPN fue de 90.3%; para P vivax, el VPP fue de 77,0% y el VPN de 90.6%: para P falciparum, el VPP fue de 66,1% y el VPN de 94,3%. El índice kappa generaI fue de 1 y el de especie fue de 0,96. El límite de detección del ADN parasitario fue de 10 fg/ml. La PCR anidada detectó una muestra con infección mixta de P. vivax y P. falciparun que, por gota gruesa, dio resultado positivo para P. vivax. Se recomienda el uso de la PCR anidada como método de tamizaje en estudios epidemiológicos para la evaluación del Programa de Control Nacional de Malaria, en estudios orientados a detectar de manera temprana parasitemias en pacientes tratados cuya posible causa pueda ser la resistencia a los antimaláricos y como prueba de referencia para evaluar nuevos métodos diagnósticos.

  8. PCR

    African Journals Online (AJOL)

    Elham

    2013-07-03

    Jul 3, 2013 ... was constructed with competitive strategy by PCR-cloning technique and the limitation range was determined. The PCR products of MTB and IAC were 245 and 660 bp, respectively on .... products' differentiation was easy.

  9. Central nervous system abnormalities on midline facial defects with hypertelorism detected by magnetic resonance image and computed tomography Anomalias de sistema nervoso central em defeitos de linha média facial com hipertelorismo detectados por ressonância magnética e tomografia computadorizada

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    Vera Lúcia Gil-da-Silva-Lopes

    2006-12-01

    Full Text Available The aim of this study were to describe and to compare structural central nervous system (CNS anomalies detected by magnetic resonance image (MRI and computed tomography (CT in individuals affected by midline facial defects with hypertelorism (MFDH isolated or associated with multiple congenital anomalies (MCA. The investigation protocol included dysmorphological examination, skull and facial X-rays, brain CT and/or MRI. We studied 24 individuals, 12 of them had an isolated form (Group I and the others, MCA with unknown etiology (Group II. There was no significative difference between Group I and II and the results are presented in set. In addition to the several CNS anomalies previously described, MRI (n=18 was useful for detection of neuronal migration errors. These data suggested that structural CNS anomalies and MFDH seem to have an intrinsic embryological relationship, which should be taken in account during the clinical follow-up.Este estudo objetivou descrever e comparar as anomalias estruturais do sistema nervoso central (SNC detectadas por meio de ressonância magnética (RM e tomografia computadorizada (TC de crânio em indivíduos com defeitos de linha média facial com hipertelorismo (DLMFH isolados ou associados a anomalias congênitas múltiplas (ACM. O protocolo de investigação incluiu exame dismorfológico, RX de crânio e face, CT e RM de crânio. Foram estudados 24 indivíduos, sendo que 12 apresentavam a forma isolada (Grupo I e os demais, DLMFH com ACM de etiologia não esclarecida (Grupo II. Não houve diferença entre os dois grupos e os resultados foram agrupados. Além de várias anomalias de SNC já descritas, a RM foi útil para detecção de erros de migração neuronal. Os dados sugerem que as alterações estruturais de SNC e os DLMFH têm relação embriológica, o que deve ser levado em conta durante o seguimento clínico.

  10. Improved diagnosis of central nervous system tuberculosis by MPB64-Target PCR Diagnóstico da tuberculose do sistema nervoso central por MPB64-Target PCR

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    Dil-Afroze

    2008-06-01

    óstico laboratorial rápido é de importância vital considerando que o espectro da doença é amplo e as anormalidades do liquor são muito variáveis. Considerando que a hipersensibilidade tardia é a resposta imune fundamental, a carga bacteriana é muito baixa. Os métodos bacteriológicos convencionais raramente detectam Mycobacterium tuberculosis no liquor e são de uso limitado para diagnóstico da meningite tuberculosa (TBM. O presente estudo duplo-cego objetivou a análise molecular da tuberculose do CNS através de um PCR desenvolvido in-house direcionado para a amplificação de uma seqüência de nucleotídios de 240pb que codificam a proteína MPB64 especifica de Mycobacterium tuberculosis. Baseando-se em critérios clínicos, selecionou-se 47 pacientes com tuberculose do CNS e um grupo controle de 10 pacientes com lesões não-tuberculosas no CNS. As análises foram divididas em três grupos: um grupo de 27 pacientes com TBM, um segundo grupo com 20 pacientes com tuberculomas intracraniais e um terceiro grupo de 10 pacientes com lesões não-tuberculosas no CNS (controles. O PCR não forneceu nenhum resultado falso-positivo, com 100% de especificidade. Em todos os três grupos de estudo, os resultados das análises de rotina do liquor por histologia, química e baciloscopia e também cultura foram negativos em todos os casos. No primeiro grupo de pacientes com TBM, PCR foi positivo em 21/27 pacientes (sensibilidade de 77,7%. No segundo grupo de pacientes com tuberculomas intracraniais, 6/20 foram positivos (sensibilidade de 30%. Nenhum dos pacientes do grupo controle foi positivo (100% de especificidade. Dessa forma, o PCR mostrou-se mais sensível que os métodos convencionais no diagnóstico de casos suspeitos de meningite tuberculosa.

  11. Determinación y cuantificación de bacterias acidolácticas por PCR en tiempo real

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    Vienvilay Phandanouvong L

    2010-04-01

    Full Text Available Objetivo. Establecer un ensayo de PCR-TR para determinar el tamaño poblacional y la composición de bacterias totales y de ácido lácticas, en particular las pertenecientes a los géneros Lactobacillus y Bifidobacterium. Materiales y métodos. La especificidad de los iniciadores fue verificada utilizando la técnica de PCR convencional. Diluciones de 101 a 10- 4 ng/μl de ADN fueron preparadas a partir de cada cultivo microbiano y utilizadas en las curvas de calibración. La temperatura de disociación y la eficiencia de cada reacción de PCR-TR se determinaron con el software del iQCycler (Bio Rad®, versión 3.1. Resultados. Los juegos de iniciadores utilizados resultaron específicos para cada grupo microbiano, sin detectar reacción cruzada. Las eficiencias de las reacciones de la PCR-TR para bacterias totales, acidolácticas, Lactobacillus y Bifidobacterium fueron 104.4%, 98.1%, 113.3% y 103.3%, respectivamente. Conclusiones. Al obtener reacciones específicas y eficiencias cercanas al 100%, es posible cuantificar las poblaciones bacterianas totales, acidolácticas, Lactobacillus y Bifidobacterium con una alta especificidad. Por lo tanto, la técnica de PCRTR puede utilizarse para monitorear cambios poblacionales bacterianos en ambientes como el tracto gastrointestinal de pollos de engorde, donde las bacterias acidolácticas, Lactobacillus y Bifidobacterium son habitantes comunes.

  12. Detection and quantification of Roundup Ready® soybean residues in sausage samples by conventional and real-time PCR Detecção e quantificação de resíduos de soja Roundup Ready® em amostras de salsicha por PCR convencional e PCR em tempo real

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    Francismar Corrêa Marcelino

    2008-12-01

    convencional e em tempo real têm sido as mais utilizadas para detectar e quantificar resíduos GM em alimentos altamente processados, respectivamente. A extração de DNA é um passo crítico durante o processo de análise. Alguns fatores, tais como, degradação de DNA, efeito de matriz e a presença de inibidores da PCR, implicam que um limite de detecção ou quantificação estabelecido para um determinado método é restrito à matriz usada durante validação e não pode ser estendido a qualquer outra matriz fora do escopo do método. No Brasil, amostras de salsicha constituíram a principal classe de produtos, nas quais resíduos de soja Roundup Ready® (RR foram detectados. Então a validação de metodologias para detecção e quantificação destes resíduos é urgente. Amostras de salsicha foram submetidas a dois diferentes métodos de extração de DNA: Wizard modificado e CTAB. O rendimento e a qualidade foram comparados em ambos os métodos. As amostras foram analisadas por PCR convencional e em tempo real para detecção e quantificação de resíduos de soja RR. Pelo menos 200 ng de DNA total da amostra de salsicha foram necessários para garantir uma quantificação confiável. Reações contendo concentrações de DNA abaixo deste limite apresentaram grande variação nos valores de porcentagem de GM esperados. Na PCR convencional, o limite de detecção variou de 1,0 a 500 ng, dependendo do conteúdo de soja GM na amostra. A precisão, performance e linearidade foram relativamente altas, indicando que o método de análise foi satisfatório.

  13. AVALIAÇÃO DE INICIADORES E PROTOCOLO PARA O DIAGNÓSTICO DA PANCITOPENIA TROPICAL CANINA POR PCR

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    Daniel C. L. Linhares

    2006-10-01

    Full Text Available Este trabalho foi desenvolvido com o objetivo de padronizar um protocolo para a reação em cadeia da polimerase (PCR e selecionar oligonucleotídeos iniciadores para a detecção específica de Ehrlichia canis, em uma única etapa de reação. Inicialmente foram obtidas seqüências depositadas no Genbank, referentes ao gene que codifica o 16S rRNA das espécies E. canis (número de acesso = AF162860, E. ewingii (U96436, E. platys (AF1567844, E. chaffeensis, (U86665, E. phagocytophila genogrupo (U02521, E. bovis (AF294789 e E. risticii (M21290, as quais foram submetidas ao alinhamento genético para a construção dos iniciadores. Do alinhamento foi selecionado, a partir de uma região semiconservada, um iniciador específico para E. canis, designado EBR1 (5’-cctctggctataggaaattg- 3’ e, de uma região conservada, um iniciador genérico EBR5 (5’-ggagtgcttaacgcgttag- 3’. Paralelamente foram obtidas amostras de sangue de dez cães que apresentavam infecção aguda por E. canis, confirmado pela presença de mórulas intracitoplasmáticas, características da riquétsia, em células mononucleares sangüíneas. O DNA genômico extraído dessas amostras foi utilizado para a avaliação da reação de PCR, empregando-se um protocolo adaptado de outros autores e o par de oligos EBR1/EBR5, selecionado neste trabalho. A reação de PCR apresentou resultados positivos para os 10 isolados de E. canis, amplificando o fragmento esperado de 765 pares de bases do gene 16S rRNA. Resultados negativos verificados nas reações de PCR para amostras de DNA genômico de Babesia canis, Hepatozoon canis, Haemobartonella sp., Trypanosoma evansi e do hospedeiro (Canis familiaris livre de infecção indicaram a segurança do método quanto à especificidade para a discriminação de E. canis. PALAVRAS-CHAVE: Cães, Ehrlichia canis, erliquiose canina, hemoparasitose, reação em cadeia da polimerase

  14. Preparation of a working seed lot of BCG and quality control by PCR genotyping Preparación de un lote semilla de trabajo de BCG y control de calidad por genotipificación por PCR

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    A Trovero

    2010-02-01

    Full Text Available The bacillus Calmette-Guérin (BCG was obtained in 1920 after successive passages leading to the attenuation of a Mycobacterium bovis strain. For the following 40 years, BCG had been replicated, resulting in substrains with genotypic and phenotypic differences. Several genomic studies have compared two BCG strains, M. bovis and Mycobacterium tuberculosis, and observed that deleted regions in the different strains could be related to differences in antigenic properties. In this work, a working seed lot was obtained from a lyophilized secondary seed lot from the BCG Pasteur strain 1173 P2 and genetically characterized. The genome was analyzed by PCR directed to five regions (RD1, RD2, RD14, RD15, DU2, using the seed lot and different available strains as templates. No genetic differences were found in the fragments studied as compared to the Pasteur strain. A total of 20 passages were carried out and no differences were found in the size of the fragments amplified by PCR. In conclusion, this method allows to control a working seed lot genotypically and to assess the stability of the BCG genome.El bacilo de Calmette-Guérin (BCG se obtuvo en 1920, después de sucesivos pasajes que llevaron a la atenuación de una cepa de Mycobacterium bovis. A lo largo de los 40 años subsiguientes la cepa BCG fue replicada y surgieron subcepas con diferencias fenotípicas y genotípicas. Se realizaron varios estudios de comparación genómica de diferentes cepas de BCG, M. bovis y Mycobacterium tuberculosis, y se observó que las deleciones de regiones en las diferentes cepas podrían estar relacionadas con diferencias en las propiedades antigénicas. En este trabajo se describe la preparación y caracterización genética de un lote semilla de trabajo obtenido a partir de un lote semilla secundaria liofilizado de la cepa BCG Pasteur 1173 P2. Se analizaron por PCR cinco regiones (RD1, RD2, RD14, RD15, DU2 en el lote semilla de trabajo utilizando como control las

  15. Etiologic diagnosis of bovine infectious abortion by PCR Diagnóstico etiológico de aborto infeccioso bovino por PCR

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    Teane Milagres Augusto da Silva

    2009-12-01

    Full Text Available Infectious abortion is a significant cause of reproductive failure and economic losses in cattle. The goal of this study was to detect nucleic acids of several infectious agents known to cause abortion including Arcanobacterium pyogenes, Bovine Herpesvirus 1, Brucella abortus, Campylobacter fetus subsp. venerealis, Chlamydophila abortus, Leptospira sp., Listeria monocytogenes, Salmonella sp., Mycoplasma bovis, Mycoplasma bovigenitalium, Neospora caninum, and Tritrichomonas foetus. Tissue homogenates from 42 fetuses and paraffin-embedded tissues from 28 fetuses and 14 placentas/endometrium were included in this study. Brucella abortus was detected in 14.2% (12/84 of the samples. Salmonella sp. DNA was amplified from 2 fetuses, and there was one positive for Neospora caninum, and another for Listeria monocytogenes. This PCR-based approach resulted in identification of the etiology in 19% of samples, or 20% if considered fetal tissues only.Aborto infeccioso é uma causa significativa de falhas reprodutivas e perdas econômicas na bovinocultura. O objetivo deste estudo foi detectar ácidos nucleicos de vários agentes infecciosos reconhecidos como causadores de aborto, incluindo-se Arcanobacterium pyogenes, Herpesvirus bovino tipo 1, Brucella abortus, Campylobacter fetus subsp. venerealis, Chlamydophila abortus, Leptospira sp., Listeria monocytogenes, Salmonella sp., Mycoplasma bovis, Mycoplasma bovigenitalium, Neospora caninum e Tritrichomonas foetus. Homogenados de tecidos de 42 fetos e tecidos incluídos em parafina de 28 fetos e 14 placentas/endométrio foram incluídos neste estudo. Brucella abortus foi detectada em 14,2% (12/84 das amostras. DNA de Salmonella sp. foi amplificado de dois fetos e houve um feto positivo para Neospora caninum e outro para Listeria monocytogenes. Essa metodologia baseada em PCR resultou na identificação da etiologia em 19% das amostras ou 20% se considerados somente os tecidos fetais.

  16. Tipificación capsular mediante PCR de aislamientos de Haemophilus influenzae no tipificables por aglutinación PCR-based capsular typing of Haemophilus influenzae isolates non-typeable by agglutination

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    G. Weltman

    2005-12-01

    Full Text Available Haemophilus influenzae es reconocido como un agente patógeno responsable de infecciones localizadas y sistémicas. Se han descrito 6 tipos de polisacáridos capsulares antigénicamente distintos (a, b, c, d, e, y f que se pueden identificar por aglutinación en lámina con antisueros específicos. También existen cepas no capsuladas (NC fenotípicamente no tipificables (NT. La introducción de la vacuna conjugada produjo una marcada disminución de las enfermedades invasivas causadas por H. influenzae tipo b. En este contexto, la tipificación capsular mediante PCR es el método más apropiado para distinguir las cepas no capsuladas de las mutantes b deficientes en cápsula (b- y detectar la presencia de cepas pertenecientes a otros serotipos que no puedan ser tipificables por aglutinación. Se determinó el genotipo capsular a 38 aislamientos de Haemophilus influenzae no tipificables por aglutinación, derivados al servicio de Bacteriología Clínica del INEI-ANLIS "Dr. Carlos G. Malbrán" en el período 2002-2004. El 78,9% de los aislamientos provenían de hemocultivos y la mayor parte de ellos estaban asociados a foco respiratorio. El 100% de los aislamientos fueron identificados como H. influenzae no capsulados mediante la técnica de PCR.Haemophilus influenzae is recognized as a pathogenic agent responsible of localized and systemic infections. Six antigenically different capsular polysaccharide types have been described (a, b, c, d, e, and f which can be identified by slide agglutination with specific antisera. Besides there are non capsulated strains that cannot be typed by slide agglutination. The introduction of the conjugated vaccine produced an important reduction of invasive diseases caused by H. influenzae type b. Capsular typing by PCR is the most appropriated method for distinguishing non capsulated strains from capsule deficient type b mutants (b- and for detecting strains of other serotypes that cannot be detected by slide

  17. Diagnosis of Neospora caninum in bovine fetuses by histology, immunohistochemistry, and nested-PCR Pesquisa de Neospora caninum em fetos bovinos por histologia, imunoistoquímica e nested-PCR

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    Aline Diniz Cabral

    2009-12-01

    Full Text Available Neospora caninum, a cause of abortion and stillbirth in cattle, was studied by histology, immunohistochemistry, and nested-PCR, using primers from the Nc5 region of the genomic DNA (PCR PLUS and primers from the ITS1 region of the ribosomal DNA (PCR JB. A total of 105 fetal samples sent to the Centro de Pesquisa e Desenvolvimento de Sanidade Animal do Instituto Biológico from January 2006 to May 2008 were examined for evidence of N. caninum. Histological examination revealed 71.4% with non-suppurative inflammation in the heart, lung, liver, kidney, placenta, and brain. Immunohistochemistry detected infections in 8.6% of the samples, mainly in the brain, placenta, and heart. Nested-PCR JB revealed 6.7% with infections, while nested-PCR PLUS returned 20.9% positive results, mainly in brain and placenta, and in the pooled liver and heart. Kappa statistics demonstrated little agreement among the three techniques. The three methods are complementary, since they have distinct diagnostic characteristics and were combined to give a positivity rate of 24.8%.Pesquisou-se Neospora caninum como causador de abortamento e natimortalidade em bovinos, por meio de exame histopatológico (hematoxilina-eosina, imunoistoquímica (IHQ e nested-PCR, utilizando primers da região Nc5 do DNA genômico (PCR PLUS e primers da região ITS1 do DNA ribossomal (PCR JB. Foram avaliadas 105 amostras de abortamento bovino entre janeiro de 2006 a maio de 2008, encaminhadas ao Centro de Pesquisa e Desenvolvimento de Sanidade Animal do Instituto Biológico para diagnóstico diferencial de causas infecciosas. Observou-se em 71,4% das amostras lesões histológicas (HE caracterizadas pela presença de células inflamatórias mononucleares no coração, pulmão, fígado, rim, placenta e cérebro. A IHQ detectou 8,57% de positividade, sendo maior no cérebro, placenta e coração. A nested-PCR JB revelou 6,66% de casos positivos, enquanto que a nested-PCR PLUS apresentou maior taxa

  18. RELACIÓN ENTRE LA ANEMIA GESTACIONAL EN LA ALTURA DETECTADO EN EL I TRIMESTRE Y LOS RESULTADOS PERINATALES. HOSPITAL DANIEL ALCIDES CARRIÓN PASCO. AÑO 2015

    OpenAIRE

    Rios Bernardo, Cledy Ruperta

    2017-01-01

    Objetivo: Determinar la relación entre la anemia gestacional en la altura detectado en el I trimestre y los resultados perinatales. Hospital Daniel Alcides Carrión – Pasco. Año 2015. Método de Estudio: Deductivo; diseño descriptivo de nivel relacional, tipo retrospectivo, de cohorte transversal; la población estudiada estuvo constituida por 44 gestantes. Resultados: 1. La edad más frecuente 18 años, el 86% procedente de la zonas distritales, con estudios de secundaria en el 64% y 73% co...

  19. Prevalencia y detección por PCR anidada de Anaplasma marginale en bovinos y garrapatas en la zona central del Litoral ecuatoriano

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    Ariel Escobar Troya

    2015-09-01

    Full Text Available La bacteria que provoca la anaplasmosis en bovinos se conoce como Anaplasma marginale, es potencialmente transmitida de forma biológica por garrapatas, moscas y fómites o mediante sangre infectada como consecuencia del uso incorrecto de herramientas quirúrgicas. Hasta la fecha, Ecuador carece de estudios actualizados sobre procedimientos eficientes para el diagnóstico específico y, erradicación de A. marginale a través del control de estos insectos, por lo que resulta de gran importancia desarrollar e implementar trabajos relacionados con el uso de esta herramienta molecular, para la detección de la bacteria en vectores de transmisión que provocan la enfermedad en bovinos. En este trabajo, se extrajo ADN eficazmente con el método de Salting Out. Un total de 255 muestras fueron analizadas por PCR anidada, distribuidas del siguiente modo|108| Riphicephalus (Boophilus microplus, |85| bovinos, de estos el 13.46% y 85.48% dieron positivas para la rickettsia, las muestras de Amblyomma spp. |62| todas fueron negativas. El índice de concordancia Kappa |total de bovinos infestados frente a Riphicephalus (Boophilus microplus|, no fue significativo (0.28. Subsiguientemente, se determinó por χ2 (p=0.66 que la presencia o ausencia de la enfermedad es independiente del lugar de donde proviene el bovino.

  20. PCR detection of four periodontopathogens from subgingival clinical samples Detecção por PCR de quatro periodontopatógenos de pacientes com doença periodontal e de indivíduos sadios

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    Mario Julio Avila-Campos

    2003-04-01

    Full Text Available In this study, A. actinomycetemcomitans, B. forsythus, P. gingivalis and F. nucleatum were identified from subgingival plaque from 50 periodontal patients and 50 healthy subjects. Subgingival clinical samples were collected with sterilized paper points and transported in VMGA III. From all the diluted clinical samples (1:10, DNA was obtained by boiling, and after centrifugation the supernatant was used as template. Specific primers for each bacterial species were used in PCR. PCR amplification was sensitive to identify these organisms. PCR products from each species showed a single band and can be used to identify periodontal organisms from clinical specimens. PCR detection odds ratio values for A. actinomycetemcomitans and B. forsythus were significantly associated with disease showing a higher OR values for B. forsythus (2.97, 95% CI 1.88 - 4.70. These results suggest a strong association among the studied species and the periodontal lesion.Em nosso estudo quatro periodontopatógenos foram isolados e identificados de placas subgengivais de 50 pacientes com doença periodontal e de 50 indivíduos sadios. As placas subgengivais foram coletadas com pontas de papel e transportadas em VMGA III. Foram realizadas diluições seriadas das amostras clínicas (1:10, e os DNA foram obtidos por fervura. Iniciadores específicos para cada bactéria foram usados no PCR. As amplificações mostraram-se sensíveis na identificação de A. actinomycetemcomitans, B. forsythus, P. gingivalis e F. nucleatum. As reações de PCR produziram bandas específicas para cada espécie e podem ser usadas na identificação desses organismos periodontais diretamente das amostras clínicas. Os valores de odds ratio para a detecção de A. actinomycetemcomitans e B. forsythus foram significativamente associados com a doença periodontal mostrando altos valores de OR para B. forsythus (2,97, 95% CI 1,88 - 4,70. Esses resultados sugerem uma forte associação entre os

  1. Diseño y evaluación de tres oligonucleótidos para la detección de Leishmania por PCR

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    Omar Cáceres Rey

    2002-07-01

    Full Text Available La leishmaniasis afecta la salud pública en 88 países del mundo y representa un serio obstáculo para su desarrollo socioeconómico. Los métodos para diagnosticar la enfermedad toman tiempo y muchas veces son traumáticos para el paciente. Objetivo: Se aplicó PCR como método alternativo para el diagnóstico rápido de esta enfermedad. Materiales y métodos: A diferentes especies de Leishmania se les extrajo ADN genómico. Se diseñaron tres oligonucleótidos (LEISH 1, LEISH 2 y LEISH 3 dirigidos al extremo carboxilo terminal de la histona H2B de Leishmania (V. peruviana cuya secuencia parcial sirvió para diseñar dichos oligos, los cuales fueron probados en un sistema de PCR. Resultados: Los oligonucleótidos amplificaron exitosamente regiones de 123 pb (LEISH 1 / LEISH 2 y 139 pb (LEISH 1 / LEISH 3 de esta secuencia parcial utilizada. La sensibilidad del PCR fue de hasta 1 fg de ADN purificado de L. (V. peruviana y de 2 parásitos cuando se realizó la técnica de manera directa. La especificidad fue 100% (solo reconoció a Leishmania y no a Trypanosoma ni a humano. Los oligonucleótidos diseñados también amplificaron todas las especies de Leishmania evaluadas. Conclusión: El sistema de PCR diseñado puede ser aplicado en la detección del parásito a partir de cualquier tipo de muestra convirtiéndose en un método alternativo de diagnóstico de la enfermedad por su rapidez, especificidad y sensibilidad.

  2. Detección por PCR de Colletotrichum lindemuthianum en cultivos y semillas de frijol en Antioquia, Colombia

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    Leonardo Martínez Pacheco

    2014-12-01

    Full Text Available Colletotrichum lindemuthianum, agente causal de la antracnosis del frijol, es uno de los patógenos más limitantes en la producción de este cultivo. La detección y correcta identificación de este hongo resulta fundamental para el manejo de la enfermedad, siendo las pruebas moleculares alternativas rápidas y sensibles para este fin. Mediante la técnica de PCR se evaluaron cuatro juegos de cebadores (CY1/CY2, CD1/CD2, ClF4/ITS4 y ClF432/ClR533 para la detección de C. lindemuthianum a partir de tejidos foliares, de vainas y de semillas procedentes de cultivos de frijol de Antioquia, Colombia. Los resultados indicaron que el par CD1/CD2, dirigido al pseudogen de permeasa de hierro Ftr1, fue el más efectivo para detectar el hongo en tejidos y semillas de frijol, así como para identificar aislamientos en cultivos microbiológicos. Para los cebadores CY1/CY2, dirigidos a los ITS del rDNA, se recomienda un esquema de PCR-RFLPs con MseI (=Tru1I para la diferenciación con las especies C. orbiculare y C. trifolii. Estos cebadores generaron resultados consistentes cuando se utilizaron en combinación con ITS1 (ITS1/CY2 e ITS4 (CY1/ITS4. Finalmente, los cebadores ClF4/ITS4 resultaron en amplificaciones inespecíficas y ClF432/ClR533 en fragmentos de difícil resolución en electroforesis de agarosa. Este estudio servirá de apoyo para los programas de certificación de semilla y mejoramiento genético de frijol.

  3. Caracterização da comunidade bacteriana endofítica de citros por isolamento, PCR específico e DGGE Characterization of the endophytic bacterial community from citrus by isolation, specific PCR and DGGE

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    Paulo Teixeira Lacava

    2006-04-01

    Full Text Available O objetivo deste trabalho foi caracterizar a comunidade bacteriana endofítica de plantas assintomáticas (escapes e afetadas pela clorose variegada dos citros (CVC por meio de isolamento em meio de cultura, técnica de gradiente desnaturante em gel de eletroforese (DGGE e detecção de Methylobacterium mesophilicum e Xyllela fastidiosa por meio de PCR específico, para estudar esta comunidade e sua relação com a ocorrência da CVC. A análise da comunidade bacteriana via DGGE permitiu a detecção de X. fastidiosa, bem como Klebsiella sp. e Acinetobacter sp. como endófitos de citros. Foram observados também Curtobacterium sp., Pseudomonas sp., Enterobacter sp. e Bacillus spp. Utilizando primers específicos, Methylobacterium mesophilicum e X. fastidiosa também foram observadas, reforçando hipóteses de que estas bactérias podem estar interagindo no interior da planta hospedeira.The aim of this work was to characterize endophytic bacterial community of assintomatic (escape and Citrus Variegated Chlorosis (CVC-affected citrus plants using isolation in culture medium, denaturing gradient gel electrophoresis (DGGE technique and Methylobacterium mesophilicum as well as Xylella fastidiosa specific PCR, allowing to assess this community and its interactions with CVC. The study of bacterial community by DGGE analysis allowed the detection of X. fastidiosa, as well as Klebsiella sp. e Acinetobacter sp., which were not detected previously. Curtobacterium sp., Pseudomonas sp., Enterobacter sp. and Bacillus spp. were also observed as endophyte in citrus plants. Using specific primers Methylobacterium mesophilicum and X. fastidiosa were observed, reinforcing that these bacteria could interact inside the host plant.

  4. Análisis por LSSP-PCR de la variabilidad genética de Trypanosoma cruzi en sangre y órganos de ratones.

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    Ana María Mejía

    2005-03-01

    Full Text Available Introducción. La enfermedad de Chagas, causada por Trypanosoma cruzi, presenta un curso clínico variable que oscila desde casos asintomáticos a casos crónicos. T. cruzi tiene una estructura clonal y las cepas infectivas son a menudo multiclonales. La variabilidad genética de T. cruzi puede ser un determinante para el tropismo diferencial a tejidos y, consecuentemente, para las formas clínicas de la enfermedad. Objetivo. Caracterizar genéticamente los parásitos de sangre y órganos de ratones infectados con dos cepas colombianas de T. cruzi. Materiales y métodos. Se infectaron ratones con dos cepas colombianas de T. cruzi con el fin de determinar la infección en sangre y órganos. Para esto, se evaluó la sensibilidad de tres marcadores moleculares diferentes, y se determinó la variabilidad genética de los clones por la técnica de reacción en cadena de la polimerasa de baja astringencia con un único iniciador específico (LSSP-PCR, utilizando el marcador del ADN del cinetoplasto (kADN. Los perfiles de bandas obtenidos con la LSSP-PCR se analizaron por el método de neighbor-joining. Resultados y conclusiones. Nuestros resultados confirmaron la presencia de los dos grupos de T. cruzi en las cepas y el carácter policlonal de éstas. El marcador más sensible fue el kADN y el órgano más afectado, el corazón. Se encontraron diferencias genéticas entre los clones presentes en la sangre y los órganos de los ratones infectados. En conclusión, estos resultados apoyan el uso de la LSSP-PCR para el entendimiento de la epidemiología de la enfermedad de Chagas.

  5. Definición de las condiciones de temperatura y almacenamiento adecuadas en la detección de ADN de Leishmania por PCR en flebotominos.

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    Olga L. Cabrera

    2002-09-01

    Full Text Available Para estudios epidemiológicos y programas de control de las leishmaniasis es importante la determinación taxonómica del insecto vector y del agente etiológico causante de esta enfermedad. Por su eficacia y sensibilidad, la utilización de técnicas moleculares, como la reacción en cadena de la polimerasa (PCR ha permitido avances en entomología médica. La metodología tradicional usada para la búsqueda de infección en los flebótomos es un método dispendioso que requiere mucho tiempo y capacitación para emitir un diagnóstico acertado. En el presente trabajo se evaluaron las condiciones y prácticas adecuadas para el almacenamiento de flebótomos con el propósito de aplicar la PCR. Hembras de Lutzomyia longipalpis de la colonia del Instituto Nacional de Salud se infectaron experimentalmente con una cepa de Leishmania chagasi del valle alto del río Magdalena (Quipile, Cundinamarca. Los insectos infectados se preservaron en tres soluciones: etanol al 100%, etanol al 70% y en buffer Tris- EDTA (TE; submuestras de cada una se almacenaron a -80 °C, -20 °C y a temperatura ambiente. Para determinar los porcentajes de infección, algunas de las hembras se disecaron para buscar las formas flageladas al microscopio. La extracción del KADN se realizó con Chelex 100. Para la amplificación se utilizaron los iniciadores OL1 y OL2 de Leishmania con electroforesis en geles de agarosa al 1%. En cada una de las condiciones descritas se logró la amplificación de un fragmento de »120 pb, correspondiente a Leishmania spp. Estos resultados muestran la ventaja de incorporar como técnica de rutina la PCR para detectar flebótomos infectados de zonas endémicas de leishmaniasis visceral. Por costo-efectividad, se concluyó que las muestras entomológicas para estudios de incriminación vectorial utilizando la PCR, se pueden preservar a temperatura ambiente en etanol al 70%.

  6. Residual tissue post splenectomy detected by splenic scintillography with erythrocytes damaged by heat; Tejido residual postesplenectomia detectado por centellografia esplenica con eritrocitos danados por calor

    Energy Technology Data Exchange (ETDEWEB)

    Rivera B, B; Garcia C, E S; Garcia O, J R [Centro Medico ABC, Departamento de Medicina Nuclear, Mexico, D.F. (Mexico)

    2005-07-01

    Feminine of 26 years old with diagnostic of purple thrombocytopenic idiopathic to those 4 years of age, tried with steroids and splenectomy at 11 years old. Pathway practically asymptomatic until 4 months ago she had presented asthenia, adynamia and general uneasiness, with platelet figures of 40,000 plat/microliter. It was carried out scintillographic study with damaged erythrocytes for post surgical remainder search. Its were took two-dimensional images and tomography by single photon emission (SPECT), being knitted splenic residual in area of anatomical projection of the spleen. (Author)

  7. Determinación del umbral de detección de Pseudococcus viburni (Hemiptera: Pseudococcidae por PCR Determination of the detection threshold of Pseudococcus viburni (Hemiptera: Pseudococcidae by PCR

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    Diana Vera

    2012-06-01

    Full Text Available La cochinilla harinosa de los frutales Pseudococcus viburni (Signoret es una plaga cuarentenaria, presente en el Alto Valle de Río Negro y Neuquén, Argentina. Su detección durante la fiscalización aduanera, aun en los estados inmaduros, provoca rechazos de la fruta fresca argentina con destino a los mercados internacionales. Las técnicas actuales de identificación de pseudocóccidos y otros cocoideos implican la realización de preparados microscópicos que requieren varios días. Por esto, la disminución de los tiempos de identificación es importante sobre todo en las tareas de fiscalización. En este trabajo, se determinó el umbral de detección específica de P. viburni mediante PCR, como así también, la implementación de un método rápido de extracción de ADN mediante DNAzolT. Insectos de diferentes estados de desarrollo (huevo, ninfas (tres estados ninfales y adulto, conservados en etanol pro análisis a -20 ºC, provenientes de montes frutales del Alto Valle, Argentina, fueron procesados según el protocolo del fabricante y se logró obtener ADN de buena calidad y concentración. Una alícuota del mismo fue utilizado como templado para una reacción de PCR usando primers específicos para P. viburni, registrados en bibliografía y como control positivo ADN de P. viburni de colección entomológica. Los primers utilizados y sus secuencias son A4 (5'-cccgcggccgttctctcttt-3' y A5 (5'-atatgttgtgcatagttgtgtgtgcgc-3', diseñados por Beuning et al. (1999. La amplificación generó una banda de peso molecular esperado de 650 pb. en gel de agarosa al 1.5% en todos los estadios, se determinó como límite de detección el estado de huevo. Esta técnica constituye una detección específica de P. viburni en un lapso máximo de 48 h.The obscure mealybug Pseudococcus viburni (Signoret is a quarantine pest present in the Upper Valley of Río Negro and Neuquén, Argentina. The detection of any growth stage of the mealybug in quarantine

  8. Hipotiroidismo congénito secundario a hipoplasia tiroidea detectado en edad adulta

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    Paula Monti

    2013-04-01

    Full Text Available La ubicación anatómica de la glándula tiroidea y su biosíntesis hormonal están reguladas por la expresión de ciertos genes, cuya alteración puede conducir a las denominadas disgenesias tiroideas: agenesia, ectopía e hipoplasia, así como a las variantes dishormonogenéticas. Se presenta el caso de una paciente con retraso mental y diagnóstico de hipotiroidismo realizado en la edad adulta. Las determinaciones bioquímicas confirmaron el diagnóstico de hipotiroidismo no autoinmune. Este caso representa la evolución prolongada de una hipofunción tiroidea, que cursó en forma solapada y no diagnosticada durante 53 años de vida, con secuelas relevantes de esta deficiencia al momento del diagnóstico. La terapia exógena logró mejorías evidentes en la signo sintomatología, pero no revirtió el presunto daño neurológico atribuible a la falta de hormona tiroidea necesaria durante el desarrollo fetal. En la necropsia realizada se encontró escaso tejido tiroideo cervical correspondiente a hipoplasia tiroidea eutópica. El hallazgo de un remanente tiroideo menor a 1 cm permite explicar la supervivencia de la paciente hasta una edad avanzada.

  9. A frequência do HPV na mucosa oral normal de indivíduos sadios por meio da PCR The frequency of human papillomavirus findings in normal oral mucosa of healthy people by PCR

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    David Esquenazi

    2010-02-01

    Full Text Available Os papilomavírus humanos (HPV são DNA vírus pertencentes à família papilomaviridae com grupos de baixo e alto risco que infectam a pele e a mucosa podendo induzir a formação de tumores epiteliais benignos e malignos. Na mucosa oral, estes vírus têm sido associados a papilomas orais, hiperplasias epiteliais focais, leucoplasias e neoplasias orais. OBJETIVO: Estudar a frequência do HPV em mucosa oral de indivíduos normais. MATERIAL E MÉTODO: Trabalho prospectivo em coorte transversal. Participaram desse estudo 100 indivíduos voluntários, faixa etária de 20 a 31 anos, estudantes universitários, sem história, queixas ou lesões visíveis ao exame físico de cavidade oral e orofaringe. Foram submetidos a questionário com perguntas referentes à epidemiologia da infecção pelo HPV. Foi colhido material de mucosa oral por raspado com escova e analisado pelo PCR. RESULTADOS: Os resultados mostraram ausência de HPV em todas as amostras. CONCLUSÃO: Parece ter havido participação do alto nível socioeconômico com alimentação rica em carotenoides e vitamina C, baixo consumo tabágico e etílico e comportamento heterossexual predominantemente monogâmico com uso regular de preservativos.The human papillomavirus (HPV is a DNA virus, which belongs to papillomaviridae family, being of low and high risk, which infect the skin and mucous membranes and can induce benign and malign tumor formation. In the oral mucosa they have been associated with oral papilloma, focal epithelial hyperplasia, leucoplakia and oral neoplasia. AIM: to study the frequency of HPV finding in oral mucosa of normal people. MATERIALS AND METHODS: Prospective study, cross-sectional cohort. One hundred volunteers, young adults, healthy, aged between 20 and 31 years, university students with no history, no complains, without oral or oropharyngeal lesions. They were submitted to a questionnaire with questions regarding HPV infection epidemiology. The samples were

  10. Genital and extra-genital screening for gonorrhoea using the BD Probetec ET system with an in-house PCR method targeting the porA pseudogene as confirmatory test

    DEFF Research Database (Denmark)

    Skovgaard, Sissel; Larsen, Helle Kiellberg; Sand, Carsten

    2012-01-01

    for Chlamydia trachomatis testing were also examined for GC on the BD Viper™ platform using the BD Probetec ET system. In order to avoid false-positive results all GC BD reactive samples were re-tested using a PCR method with the porA pseudogene as target. Using this method we screened 170% more samples for GC...

  11. ANÁLISIS GENÓMICO DE PARVOVIRUS CANINO POR PCR - RFLP A PARTIR DE AISLAMIENTOS DE CASOS CLÍNICOS SINTOMÁTICOS TOMADOS EN BOGOTÁ - COLOMBIA -ESTUDIO PRELIMINAR-

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    Ángela Castillo

    2001-12-01

    Full Text Available Con el objetivo de detemúnar el genotipo del parvovirus canino (CPV circulante en Bogotá, se analizaron 56 muestras tomadas a conveniencia, de materia fecal y sangre de perros menores a un año con gastroenteritis hemorrágica presuntiva de parvovirosis, por reacción en cadena de la polirnerasa - huella genórnica (PCR - RFLP. Se obtuvieron fragmentos amplificados de DNA de 2.2 Kb por PCR de la cepa vacunal de CPV-2 y de cuatro muestras de heces, no se obtuvieron amplificados de las muestras de sangre. Los productos amplificados fueron digeridos por la enzima de restricción Rsa I pemútiendo la identificación de los genotipos CPV-2a y CPV-2b así como su diferenciación con el parvovirus tipo - 2 (CPV-2.

  12. Detecção de Brucella abortus em tecidos bovinos utilizando ensaios de PCR e qPCR¹

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    Marrielen A.B. Caitano

    2014-06-01

    Full Text Available Objetivou-se no presente estudo avaliar as técnicas reação em cadeia da polimerase (PCR e PCR em Tempo Real (qPCR para detectar Brucella abortus, a partir de tecidos bovinos com lesões sugestivas de brucelose. Para isto, 21 fragmentos de tecidos bovinos coletados em abatedouros de Mato Grosso do Sul foram processados e submetidos ao cultivo microbiológico e extração do DNA genômico para realização das reações de PCR e qPCR. No cultivo microbiológico, oito amostras apresentaram crescimento bacteriano e cinco foram confirmadas como B. abortus por PCR. Diretamente das amostras de tecido, DNA do gênero Brucella (oligonucleotídeos IS711 foi detectado em 13 (61,9% amostras de tecido e 17 (81% amostras de homogeneizado. Já com os oligonucleotídeos espécie-específicos BruAb2_0168F e BruAb2_0168R, 14 (66% amostras de tecido e 18 (85,7% amostras de homogeneizado foram amplificadas. Seis amostras positivas na PCR espécie-específica foram sequenciadas e o best hit na análise BLASTn foi B. abortus. Na qPCR, 21 (100% amostras de tecidos e 19 (90,5% amostras de homogeneizado foram positivas para B. abortus. Dez amostras de DNA de sangue bovino de rebanho certificado livre foram utilizadas como controle negativo nas análises de PCR e qPCR utilizando-se os oligonucleotídeos BruAb2_0168F e BruAb2_0168R. Na PCR nenhuma amostra amplificou, enquanto que na qPCR 2 (20% amplificaram. Conclui-se que as duas técnicas detectam a presença de B. abortus diretamente de tecidos e homogeneizados, porém a qPCR apresentou maior sensibilidade. Os resultados obtidos indicam que a qPCR pode representar uma alternativa rápida e precisa para a detecção de B. abortus diretamente de tecidos, e ser utilizada em programas de vigilância sanitária, por apresentar sensibilidade e especificidade satisfatórias.

  13. Atypical rotavirus among diarrhoeic children living in Belém, Brazil Rotavírus atípicos detectados em crianças diarréicas, em Belém, Brasil

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    Yvone B. Gabbay

    1989-03-01

    Full Text Available Atypical rotaviruses were detected in faeces from two diarrhoeic children living in Belém, Pará, Brazil. Rotavirus particles were detected by electron microscopy and the RNA electrophoresis showed patterns which were compatible with group C rotaviruses. Tests for the presence of group A antigen by enzyme-linked-immunosorbent assay (ELISA were negative. The two children had three successive rotavirus infection and in both cases the atypical strains were excreted at the time of the third infection, causing a mild and short-lasting disease.Rotavírus atípicos foram detectados nas fezes de duas crianças diarreícas residentes em Belém, Brasil. Partículas de rotavírus foram visualizadas por microscopia eletrônica nos espécimes fecais de ambos os pacientes, tendo a eletroforese do ácido ribonucleico (ARN exibido padrões compatíveis com rotavírus do grupo C. Testes imunoenzimáticos (ELISA foram negativos quanto à presença de antígenos do grupo A. As duas crianças apresentaram três infecções sucessivas por esse agente, sendo que, em ambos os casos, os rotavírus atípicos foram excretados por ocasião da terceira infecção, produzindo sintomas brandos e de pouca duração.

  14. PCR associated with hybridization with DNA radioactive probes for diagnosis of asymptomatic infection caused by Leishmania Chagasi; PCR associado a hibridizacao com sondas radioativas de DNA para a identificacao de infeccao subclinica causada por Leishmania Chagasi

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    Andrade, Antero Silva Ribeiro de [Centro de Desenvolvimento da Tecnologia Nuclear (CDTN), Belo Horizonte, MG (Brazil); Moreno, Elizabeth Castro [Fundacao Nacional de Saude, Belo Horizonte, MG (Brazil). Coordenacao Regional de Minas Gerais; Gomes, Rosangela Fatima; Melo, Maria Norma de; Carneiro, Mariangela [Minas Gerais Univ., Belo Horizonte, MG (Brazil). Dept. de Parasitologia; Fernandes, Octavio [Fundacao Inst. Oswaldo Cruz (FIOCRUZ), Rio de Janeiro, RJ (Brazil). Dept. de Medicina Tropical

    2002-07-01

    Detection systems for diagnosis of leishmaniasis based on PCR are very promising due to their sensitivity and specificity. Secondary detection by specific radioactive DNA probes, able to type the PCR amplified products, increase the specificity and raise about tem-fold the sensitivity of the assay. The aim of this work was evaluate PCR and hybridization as a tool to identify Leishmania (Leishmania) chagasi (the specie that cause the visceral leishmaniasis in Brazil) infection in asymptomatic persons living in a endemic area. Material and Methods: A group of 226 asymptomatic individuals, living in General Carneiro (MG), was selected. Blood samples were harvested and the DNA extracted from the mononucleate cells. PCR was performed using primers addressed to the kinetoplast DNA minicircles. This protocol gives a positive reaction for all Leishmania species. The amplified products were further hybridized with cloned L.chagasi minicircles labeled with {sup 32} P. Results: were identified 111 samples PCR positive, 2 of them hybridization negative and 133 samples hybridization positive, 24 of them PCR negative. The occurrence of samples with hybridization positive and PCR negative was expected since hybridization, with DNA probes labeled with {sup 32} P, increase the sensitivity of the assay. The samples that presented positive PCR and negative hybridization were probably due the presence of other Leishmania species, likely L. (V.) braziliensis (that produce tegumentary leishmaniasis in the region), since L. (L.) chagasi cloned minicircles were used as hybridization probe. We conclude that this procedure is a valuable tool to access subclinical L. (L.) chagasi infections in epidemiological studies. (author)

  15. Caracterización, por RAPD-PCR, de aislados de Pseudomonas aeruginosa obtenidos de pacientes con fibrosis quística RAPD-PCR characterization of Pseudomonas aeruginosa strains obtained from cystic fibrosis patients

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    Maribel Ortiz-Herrera

    2004-04-01

    Full Text Available OBJETIVO: Caracterizar a las cepas de P aeruginosa aisladas de lavados broncoalveolares de pacientes con fibrosis quística a lo largo de un periodo de tres años. MATERIAL Y MÉTODOS: Estudio prospectivo, de seguimiento de una población de pacientes con fibrosis quística. Se utilizó la técnica de la amplificación del ADN empleando PCR con bajas condiciones de especificidad (Random amplified polymorphic DNA, RAPD-PCR para la amplificación del ADN de cepas de P aeruginosa aisladas de lavados broncoalveolares de cinco pacientes con fibrosis quística, provenientes del Servicio de Neumología y Cirugía del Tórax del Instituto Nacional de Pediatría de la Ciudad de México, en el periodo de junio de 1996 a junio de 2002; se establecieron los patrones de amplificación de cada aislamiento, lo que permitió la identificación precisa de todas las cepas aisladas y el estudio de la epidemiología de P aeruginosa en los pacientes seleccionados con dicha enfermedad. RESULTADOS: Se definieron 18 patrones de amplificación del ADN que permitieron identificar a cada cepa de P aeruginosa aislada en las diferentes muestras de lavado broncoalveolar; no se encontró relación entre el fenotipo de P aeruginosa (mucoide o no mucoide y el genotipo de cada aislamiento, ya que cepas con fenotipos distintos mostraron patrones de amplificación semejantes; en nuestros pacientes se identificaron cepas con patrones de amplificación distintos a partir de una misma muestra, lo que sugiere la presencia de infecciones simultáneas por más de una cepa de P aeruginosa; se demostró que dos hermanos con la enfermedad compartían cepas con genotipos semejantes, lo que sugiere una contaminación cruzada entre ambos, y se demostró el aislamiento de cepas de P aeruginosa con genotipos semejantes a lo largo de los periodos estudiados. CONCLUSIONES: La identificación mediante la caracterización genotípica de las cepas de P aeruginosa aisladas de los pacientes con

  16. DNA fingerprinting by ERIC-PCR for comparing Listeria spp. strains isolated from different sources in San Luis: Argentina Caracterización molecular por ERIC-PCR de cepas de Listeria spp. aisladas de diversos orígenes en San Luis: Argentina

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    A. Laciar

    2006-04-01

    Full Text Available In this study, a total of 24 Listeria spp. strains were analyzed. Twenty-two isolates were obtained in San Luis (Argentina from human, animal, and food samples. Two types of strains, Listeria monocytogenes CLIP 22762 and Listeria innocua CLIP 74915, were included as reference strains. All isolates were biochemically identified and characterized by serotyping, phage typing, and amplification of the flaA gene by polymerase chain reaction (PCR. Repetitive intergenic consensus (ERIC sequence-based PCR was used to generate DNA fingerprints. On the basis of ERIC-PCR fingerprints, Listeria spp. strains were divided into three major clusters matching origin of isolation. ERIC-PCR fingerprints of human and animal isolates were different from those of food isolates. In addition, groups I and II included ten L. monocytogenes strains, and only one Listeria seeligeri strain. Group III included nine L. innocua strains and four L. monocytogenes strains. Computer evaluation of ERIC-PCR fingerprints allowed discrimination between the tested serotypes 1/2b, 4b, 6a, and 6b within each major cluster. The index of discrimination calculated was 0.94. This study suggests that the ERIC-PCR technique provides an alternative method for the identification of Listeria species and the discrimination of strains within one species.En este estudio se analizaron 24 cepas de Listeria spp. De ellas, 22 fueron obtenidas en San Luis (Argentina, a partir de muestras humanas, de animales y alimentos. Se incluyeron 2 cepas de referencia Listeria monocytogenes CLIP 22762 y Listeria innocua CLIP 74915. Todos los aislamientos fueron identificados bioquímicamente y caracterizados por serotipificación, fagotipificación y detección del gen flaA por reacción en cadena de la polimerasa (PCR. Se generaron perfiles de bandas de ADN mediante la amplificación de secuencias repetitivas de consenso intergénico de enterobacterias (ERIC-PCR. De acuerdo a los resultados obtenidos por ERIC-PCR

  17. Vírus, viróides, fitoplasmas e espiroplasmas detectados em plantas ornamentais no período de 1992 a 2003.

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    Maria Amélia Vaz Aalexandre

    2005-06-01

    Full Text Available No período de 1992 a 2003 foi investigada, visando diagnóstico, a presença de vírus, viróides, fitoplasmas e espiroplasmas em 167 gêneros de plantas ornamentais, pertencentes a 64 famílias. Para o diagnóstico e a identificação desses patógenos, foram realizados ensaios biológicos, serológicos e/ou moleculares, bem como observações ao microscópio eletrônico de transmissão. Tospovirus foram detectados em 104 amostras pertencentes a 15 gêneros, incluindo flores de corte, envasadas e folhagens; Potyviridae estavam presentes em 23 gêneros, dentre esses Hyppeastrum, Lilium, Gladiolus e Dieffenbachia infectados com Potyvirus; Cucumovirus (Cucumber mosaic virus e Ilarvirus foram detectados em 14 e 5 gêneros, respectivamente; Tobamovirus e Potexvirus, que são de ocorrência freqüente no Brasil, infectaram 40,4% das orquídeas que representaram 29,3% do total de amostras recebidas; outros 7 gêneros (Badnavirus, Carlavirus, Caulimovirus, Furovirus, Nepovirus, Tobravirus e Tymovirus e duas famílias (Closteroviridae e Rhabdoviridae de fitovírus foram também detectados. Nesse período, pelo menos três novas espécies de vírus foram descritas infectando crisântemo, petúnia e caládio. Viróide foi detectado em crisântemo, fitoplasma em lisiantos e espiroplasma em prímula.

  18. Detection of Mycoplasma hyopneumoniae in lungs and nasal swabs of pigs by nested PCR Detecção de Mycoplasma hyopneumoniae em pulmões e suabes nasais de suínos por nested PCR

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    F.M.F. Silva

    2009-02-01

    Full Text Available Fifty-four samples were collected from growing and finishing pigs for the molecular diagnosis of enzootic porcine pneumonia. Nineteen lung fragments were obtained from pigs that showed signs of respiratory disease and 35 nasal swabs were obtained from clinically healthy pigs. For the detection of the bacterial genome in the samples, the nested PCR technique was used to amplify a fragment of 706bp. This fragment was subsequently cloned and sequenced. The sequence of obtained nucleotides was compared with six other sequences of Mycoplasma hyopneumoniae and 11 sequences of other bacteria available in the Genbank. To measure the sensitivity of the nested PCR, serial dilutions (10-1 to 10-15 of cloned fragments were conducted based on the concentration of 300ng. Ten lung fragments and eight nasal swabs showed positive for M. hyopneumoniae and the limit of detection was estimated to be 0.3fg DNA cloned. The sequence of nucleotides obtained showed 99.1% homology with the other sequences of M. hyopneumoniae, demonstrating that the nested PCR used in this study may provide an important diagnostic tool for the detection of this agent.Foram coletadas 54 amostras de animais em fase de crescimento e terminação para o diagnóstico molecular da pneumonia enzoótica suína. Dezenove fragmentos de pulmão foram obtidos de suínos que apresentavam sinais de doença respiratória e 35 suabes nasais foram obtidas de suínos clinicamente saudáveis. Para a detecção do genoma bacteriano nas amostras, foi utilizada a técnica de nested PCR que originou um fragmento de 706pb, o qual foi, posteriormente, clonado e sequenciado. A sequência de nucleotídeos obtida foi comparada com outras seis sequências de Mycoplasma hyopneumoniae e 11 sequências de outras bactérias disponíveis no Genbank. Para medir a sensibilidade da nested PCR, foram realizadas diluições seriadas (10-1 a 10-15 do fragmento clonado, partindo da concentração de 300ng. Dez fragmentos de pulm

  19. PCR para la confirmación de transmisión experimental de Leishmania chagasi a hámster sano por picadura de Lutzomyia longipalpis (Diptera: Psychodidae.

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    Olga L. Cabrera

    2003-06-01

    Full Text Available Se evaluó la efectividad de la PCR como herramienta en la detección de la transmisión experimental de Leishmania chagasi a hámster, Mesocricetus auratus, por picadura del insecto vector. Dos pares de hámsteres sanos y anestesiados fueron colocados en jaulas que contenían hembras de Lutzomyia longipalpis. Previamente, las hembras se infectaron experimentalmente con Leishmania chagasi y la infección se confirmó por disección en una submuestra. A los 37 y 51 días después de la exposición a los insectos infectados, las biopsias de hígado y bazo de cada hámster se sometieron a examen directo al microscopio, histopatología y PCR. El ADN se extrajo con Chelex 100®; en la amplificación se utilizó un par de iniciadores específicos para la región conservada de los minicírculos del ADN de Leishmania. El producto amplificado se separó en geles de agarosa y se visualizó bajo luz UV. En tres de las cuatro biopsias se observó una banda de 120 pares de bases, aproximadamente, correspondiente al tamaño esperado de la fracción del minicírculo. La técnica de PCR fue el único método que detectó la presencia del parásito. Estos resultados demostraron que la sensibilidad de la PCR acelera los procesos de incriminación vectorial de las especies vectoras de leishmaniasis.

  20. In vitro culture, PCR , and nested PCR for the detection of Theileria equi in horses submitted to exercise Cultivo in vitro, PCR e nested PCR na detecção de Theileria equi em eqüinos submetidos a exercícios

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    C.D. Baldani

    2008-06-01

    Full Text Available This study compared the usefulness of in vitro culture, PCR, and nested PCR for the diagnosis of Theileria equi in horses submitted to stress during exercise. Blood samples from 15 apparently healthy horses, previously conditioned to a high-speed equine treadmill, were taken prior to and after exercise. The animals were divided into two experimental groups: 30-day training schedule (G1 and 90-day training schedule (G2. Statistical analysis was performed using a chi-square test and kappa statistic was used in order to assess agreement. No significant difference was observed between samples collected at resting or after exercise. In G1, merozoites of T. equi were detected in the blood smears of four horses before in vitro culture, whereas 14 samples were positive, confirmed by culture. In G2, five and 11 horses were positive before and after culture, respectively. No PCR amplified product was observed in any of the tested animals although the PCR system based on the 16S rRNA gene of T. equi detected DNA in blood with an equivalent 8x10-5% parasitaemia. The nested PCR based on the T. equi merozoite antigen gene (EMA-1 allowed the visualization of amplified products in all the horses. Therefore, nested PCR should be considered as a means of detection of sub-clinical T. equi infections and in vitro culture could be used as a complement to other methods of diagnosis.Comparou-se a utilização do cultivo in vitro, PCR e nested PCR no diagnóstico de Theileria equi em eqüinos submetidos ao estresse induzido por exercícios. Amostras de sangue foram obtidas de 15 eqüinos submetidos a treinamento em esteira rolante de alto desempenho, sendo as amostras colhidas antes e após os exercícios. Os animais foram divididos em dois grupos experimentais: 30 dias de treinamento (G1 e 90 dias de treinamento (G2. O teste do qui-quadrado foi empregado para as análises estatísticas e o índice kappa utilizado para avaliar a concordância. Não houve diferen

  1. Comparación de métodos de extracción de ADN en tejidos parafinados y utilidad para amplificación por PCR

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    Javier Alonso Baena Del Valle

    2013-01-01

    Formalin-fixed and paraffin-embedded tissues are a source of important molecular findings in clinical and scientific, demonstrating that the DNA extracted from these is suitable for amplification by polymerase chain reaction (PCR. In this study, we tested three methods of DNA extraction, in order to compare the efficiency of these DNA for RCP amplification. Three samples were used, corresponding to a lung biopsy, endometrial curettage and lymph node, all fixed in 10% formaldehyde and embedded in paraffin. Three different methods were used for DNA extraction (extraction by salting out, modified Sambrook method and commercial kit The DNA obtained was analyzed by spectrophotometry, and gel electrophoresis was performed in 1% agarose to check if the DNA was amplifiable. PCR was performed for exon 3 of caveolin-1 gene. All methods resulted in a good product of genomic DNA, obtaining more quality and purity in the salting out and commercial kit methods. Also, we obtained amplification of the product by these two methods, without favorable results with the DNA extracted by the modified "Preparation of Genomic DNA from Mouse Tails and Other Small samples, according to Sambrook et al." The DNA obtained from FFPE can be amplified by several methods, among them, salting out extraction is an easy, effective and low toxicity for obtaining good quality DNA for PCR amplification.

  2. Transmission of Helicobacter pylori via water for human consumption. Determination by means of the molecular method using PCR; Helicobacter pylori, su transmision a traves de las aguas para el consumo humano. Determinacion por metodo molecular con la PCR

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    Amo, A. J.; Moreno, C.; Apraiz, D.; Catalan, V. [LABAQUA. Alicante (Spain)

    1998-12-31

    Helicobacter pylori is the cause of chronic gastritis, duodenal ulcers and stomach cancer. Its natural reservoirs and transmission mechanisms are still little understood. The gastro-oral route-especially vomiting and regurgitation-is one transmission path, especially in young children and babies. It has been found in some patients saliva and dental plaque, which suggests a possible oral-oral contagion route. The discovery of H. Pylori human excrement points to a faecal-oral route as the main transmission pathway via water for human consumption. It is therefore important to develop detection methods such as isolation by culture and polymerase chain reaction (PCR) to effectively monitor this pathogen in human drinking water. (Author) 30 refs.

  3. Reporte del primer caso de enfermedad de Chagas transplacentaria analizado por AP-PCR en Moniquirá, Boyacá

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    Concepción Judith Puerta

    2009-12-01

    Conclusiones. Éste es el primer caso de enfermedad de Chagas transplacentaria reportado en el municipio de Moniquirá, que demuestra que esta forma de transmisión ocurre en el país. La presencia de infección mixta por ambos grupos de T. cruzi en las muestras del recién nacido, sugiere infección mixta en la madre, con mayor prevalencia de T. cruzi I, al menos en el hemocultivo.

  4. Detecção do gene da nucleoproteína do vírus da cinomose canina por RT-PCR em urina de cães com sinais clínicos de cinomose Detection of canine distemper virus nucleoprotein gene by RT-PCR in urine of dogs with distemper clinical signs

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    C.M.S. Gebara

    2004-08-01

    Full Text Available A presença do vírus da cinomose canina (CDV foi avaliada pela reação em cadeia da polimerase, precedida de transcrição reversa (RT-PCR, em 87 amostras de urina de cães que apresentavam sinais clínicos sugestivos de cinomose. Os animais foram distribuídos em três grupos. No grupo A foram incluídos 41 cães com alterações sistêmicas; no grupo B, 37 cães com alterações neurológicas; e no grupo C, nove cães com alterações sistêmicas e neurológicas simultâneas. O grupo D (controle foi composto por 20 cães assintomáticos. Os resultados da RT-PCR foram correlacionados com a forma clínica da infecção e com as alterações hematológicas encontradas. Foi possível a amplificação parcial do gene da nucleoproteína do CDV em 41 (47,1% das 87 amostras de urina provenientes de cães com sinais clínicos sugestivos de cinomose. Todas as amostras obtidas de animais assintomáticos foram negativas na RT-PCR. Amostras positivas foram encontradas nos três grupos de animais com sinais clínicos na proporção de 51,2% (24/41, 29% (11/37 e 100% (9/9 para os grupos A, B e C, respectivamente. A leucocitose foi a alteração hematológica mais freqüente nos três grupos de cães com sinais clínicos porém, não foi possível estabelecer correlação entre o resultado da RT-PCR e as alterações hematológicas. Os resultados demonstraram que, independente da forma de apresentação clínica, a técnica da RT-PCR realizada em urina pode ser utilizada no diagnóstico ante mortem da infecção pelo CDV.The urine of 87 dogs with clinical signs suggestive of canine distemper was analyzed by RT-PCR for detection of canine distemper virus (CDV nucleoprotein gene. The samples were allotted to the following groups: group A- with 41 dogs with systemic symptoms, group B- with 37 dogs with neurological signs, and group C- with 9 dogs with simultaneous systemic and neurological clinical signs. Group D (control included 20 assymptomatic dogs. A chi2

  5. Epidemiological survey on Mycoplasma gallisepticum and M. synoviae by multiplex PCR in commercial poultry Investigação epidemiológica de Mycoplasma gallisepticum e M. synoviae por PCR Multiplex em estabelecimentos comerciais de aves

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    Marcos Roberto Buim

    2009-07-01

    Full Text Available Mycoplasmas are important avian pathogens, which cause respiratory and joint diseases that result in large economic losses in Brazilian and world-wide poultry industry. This investigation regarding the main species of mycoplasmas, Mycoplasma gallisepticum (MG and M. synoviae (MS, responsible for the above mentioned conditions, was carried out through PCR Multiplex analysis. One thousand and forty-six (1,046 samples of tracheal swabs and piped embryos were collected from 33 farms with laying hens, breeders, broilers or hatchery, located in the Brazilian states of São Paulo, Paraná and Pernambuco, where respiratory problems or drops in egg production had occurred. The MG and MS prevalence on the farms was 72.7%. These results indicated (1 high dissemination of mycoplasmas in the evaluated farms, with predominance of MS, either as single infectious agent or associated with other mycoplasmas in 20 farms (60.6%, and (2 an increase of MS and decrease of MG infection in Brazilian commercial poultry.Os Micoplasmas são importantes patógenos aviários que causam doenças respiratórias e de articulações que resultam em grandes perdas econômicas para a indústria avícola brasileira e mundial. O estudo das principais espécies de Mycoplasma, Mycoplasma gallisepticum (MG e M. synoviae (MS, responsáveis pelas doenças mencionadas acima, foram analisadas pela técnica de PCR Multiplex. Foram colhidas 1046 amostras de suabe traqueal e embriões bicados de 33 estabelecimentos de aves de postura, matrizes, frangos de corte e um incubatório, localizados nos Estados brasileiros de São Paulo, Paraná e Pernambuco, as quais apresentavam problemas respiratórios ou queda na produção de ovos. A prevalência de MS e MG nas granjas foi de 72,7%. Os resultados indicaram uma alta disseminação de Mycoplasma nas granjas avaliadas, com predominância de MS, como um único agente infeccioso ou associado com outros micoplasmas em 20 granjas (60,6%. Assim, este

  6. Detection of Toxoplasma gondii by PCR and mouse bioassay in commercial cuts of pork from experimentally infected pigs Detecção do Toxoplasma gondii por PCR e bioensaio em camundongo em cortes comerciais de carnes de suínos infectados experimentalmente

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    V.S. Tsutsui

    2007-02-01

    Full Text Available The distribution of T. gondii in commercial cuts of pork (ham, tenderloin, spareribs and arm picnic by PCR and bioassay from experimentally infected pigs, was evaluated. Eighteen mixed breed pigs were divided into two groups (G. The G1 animals (n=10 were infected with 4 x10(4 oocysts of the T. gondii VEG strain and the G2 animals (n=8 were used as control. Pigs of both groups were slaughtered at 59th day after infection, and meat samples were collected for bioassay and PCR. All animals from G1 were positive by at least one or both tests, and all control animals were negative. T. gondii was identified in pork by mouse bioassay and PCR in 27/40 (67.5% and in 9/40 (22.5% of the evaluated samples, respectively. There were no statistical differences in the distribution of tissue cysts from commercial cuts of pork by bioassay (P>0.05. However, statistical differences were observed when mouse bioassay and PCR were compared (PAvaliou-se a presença de T. gondii em cortes comerciais de carne suína (pernil, lombo, costela e paleta, por meio do bioensaio e PCR, em animais experimentalmente inoculados. Dois grupos (G foram formados. Os animais do G1 (n=10 foram inoculados com 4 x10(4 oocistos da cepa VEG e os do G2 (n=8 permaneceram como grupo-controle, não inoculado. Todos os animais foram abatidos no dia 59 após a infecção, quando foram colhidas as amostras de carne para a realização das provas de bioensaio e da PCR. Todos os suínos do G1 apresentaram-se positivos a pelo menos um dos testes de diagnóstico ou a ambos, e os do grupo-controle permaneceram negativos. Não houve diferenças significativas em relação aos tipos de cortes comerciais e à presença do parasita no bioensaio (P>0,05. O bioensaio foi capaz de detectar T. gondii em 27/40 (67,5% amostras e a PCR em 9/40 (22,5%. O estudo mostrou diferença entre o bioensaio e a PCR (P<0,01.

  7. Discriminação de sorovares de Salmonella spp. isolados de carcaças de frango por REP e ERIC-PCR e fagotipagem do sorovar Enteriditis Discrimination of Salmonella serovars isolated from chicken meat by REP and ERIC-PCR and phagotyping of Enteriditis sorovar

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    Iliana Alcocer

    2006-06-01

    Full Text Available Salmonelose é a infecção bacteriana de origem alimentar mais freqüente no Paraná, Brasil, e os surtos estão associados, principalmente, ao consumo de ovos, carne de aves e derivados. Os objetivos deste trabalho foram identificar os sorovares de Salmonella isolados de carcaças de frango e caracterizá-los molecularmente por REP e ERIC-PCR, assim como identificar os fagotipos de Salmonella Enteriditis. Dos 25 isolados de Salmonella spp. analisados, 18 foram identificados como Enteriditis, 4 como Braenderup, 2 como Worthington e 1 como infantis. Dos 18 isolados de Enteriditis, 14 foram PT4, 2 PT4a, 1 PT7 e 1 RDNC, por se tratar de colônia rugosa. REP-PCR forneceu padrão eletroforético distinto de 10 a 13 bandas distribuídas entre 120 e 2072 pb para cada sorovar diferente testado. A ERIC-PCR mostrou um padrão de 4 a 5 bandas entre 180 e 1000 pb e foi menos discriminativa quando comparada à REP-PCR. Os resultados encontrados confirmaram que a fagotipagem é uma ferramenta útil e discriminativa para o sorovar Enteriditis. Apesar do pequeno número de sorovares testados, os resultados sugerem que a REP-PCR parece ser um método atrativo a ser utilizado no futuro para a discriminação preliminar de sorovares de Salmonella.Salmonellosis is the most prevalent bacterial food-borne disease in the State of Paraná, Brazil, and the outbreaks are often associated with consumption of poultry products. The aim of this study was to serotype Salmonella strains isolated from chicken carcasses and characterize them molecularly using REP and ERIC-PCR. The phage types of Salmonella Enteriditis were also identified. Of the 25 Salmonella strains analysed, 18 were identified as Enteriditis, 4 as Braenderup, 2 as Worthington and 1 as infantis. Of the 18 Enteriditis isolates, 14 were PT4, 2 PT4a, 1 PT7 and 1 "reacted, but did not conform" - RDNC. Distinct REP-PCR profiles with 10 to 13 fragments distributed between 120 and 2072 pb were easily obtained for

  8. Molecular identification of Pseudoplatystoma sp. fish fillets by Multiplex PCR / Identificação molecular de filés de peixe Pseudoplatystoma sp. por PCR-Multiplex

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    Cátia Maria de Oliveira Lobo

    2014-08-01

    Full Text Available Nuclear and mitochondrial genes were used as molecular markers for verifying the iden-tity of fish fillets marketed as pintado (Pseudoplatystoma corruscans. Based on THE polymorphisms of nuclear DNA (RAG2, globin, and EF1 genes and mitochondrial regions (16S, we examined whether the fillets originated from inbred species of pintado or from hybrids derived from crosses between cachara (P. reticulatum and pintado. Nuclear genes from both species were detected in the analyzed fillets (n = 29. This clearly iden-tified these fish as interspecific hybrids (or F1/first filial generation of the type “cacha-pinta,” resulting from a cross between female cachara and male pintado. These results demonstrate that monitoring fish fillet trading is crucial for detecting discrepancies between the marketed species and related information declared on the label. Species that are frequently hybridized, such as pintado and cachara, require special attention. -------------------------------------------------------------------- Marcadores moleculares (PCR-Multiplex de genes nucleares e mitocondriais foram uti-lizados para verificar a identidade molecular de filés de peixe comercializados como pintado (Pseudoplatystoma corruscans com base em polimorfismos de regiões do DNA nuclear (genes RAG2, globina e EF1 e mitocondriais (16S para verificar se os filés per-tenciam a espécie pura de pintado ou se eram híbridos derivados do cruzamento entre cachara (Pseudoplatystoma reticulatum e pintado (Pseudoplatystoma corruscans. Os filés analisados (n = 29 apresentaram genes nucleares de ambas espécies P. corruscans e P. reticulatum, e desta forma, foram identificados como híbridos interespecíficos ou F1 (primeira geração filial do tipo “cachapinta” resultante do cruzamento entre uma fêmea de cachara e um macho de pintado. Estes resultados mostram que o monitora-mento da comercialização de filés de peixe é fundamental para identificar situações onde

  9. Resultado de tratamiento "fisiológico" en casos de displasia del desarrollo de la cadera (DDC detectados tardíamente

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    Néctar León Daza

    2007-01-01

    Full Text Available La maleabilidad del esqueleto del niño, que perdura a través de toda la infancia, permite que mediante la orientación de las fuerzas naturales que actúan sobre la articulación coxo-femoral, se consiga en forma ¿fisiológica¿ que vuelva a la normalidad la cadera de un niño cuya Displasia del Desarrollo de la Cadera (DDC se diagnosticó tardíamente. En esta forma se evitan los riesgos anestésico-quirúrgicos en la infancia y la ¿catastrófica¿ coxo-artrosis a la que inexorablemente conduce el manejo ortopédico-quirúrgico que en el mundo entero reciben hoy los casos de DDC detectados tardíamente.

  10. Porfirias poco frecuentes: Casos detectados en la población argentina Non frequent porphyrias in the argentinean population

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    V A Melito

    2006-12-01

    Full Text Available Las porfirias son consecuencia de fallas en el metabolismo del hemo. Se clasifican según el tipo de sintomatología clínica prevalente o el órgano donde se expresa preferencialmente la falla metabólica. En general la deficiencia enzimática está asociada a mutaciones en los genes que codifican para cada una de las enzimas. Están descritos 7 tipos de porfiria diferentes. Se transmiten por carácter autosómico dominante a excepción de la PCE, la PHE y la NPA que son recesivas. Sin embargo, están reportadas variantes homocigotas para el resto de las porfirias de pronóstico y evolución mucho más grave que la forma heterocigota. La descripción de estos casos poco frecuentes, sus tratamientos y evolución, facilitarían tanto el diagnóstico diferencial de la porfiria como el conocimiento de las posibilidades terapéuticas en cada caso. Asimismo para las porfirias heterocigotas con manifestación infantil, su identificación temprana y tratamiento aseguraría una mejor evolución minimizando los riesgos asociados. Se han diagnosticado 5 casos de porfirias agudas en niñas: 2 de PAI, 2 de PV y 1 de CPH. Entre las porfirias cutáneas se presentan 25 casos de PCT infantil, el primer caso de PHE en Argentina, 4 casos de PCE infantil y 1 en un adulto y 2 casos de PPE con compromiso hepatobiliar.The Porphyrias are a group of diseases resulting from partial deficiencies in one of the heme biosynthetic enzymes. These disorders can be classified on the basis of their clinical manifestations or according the organ where the metabolic deficiency is mainly expressed. In general this enzyme deficiency is associated with mutations in the genes which codify each enzyme. There are 7 types of Porphyrias. They are autosomal dominant disorders with the exception of PCE, PHE and NPA which are recessive. However, some rare and severe cases with recessive inheritance have also been reported. The description of these infrequent cases and their treatments and

  11. Virus isolation and molecular characterization of canine distemper virus by RT-PCR from a mature dog with multifocal encephalomyelit Isolamento e caracterização molecular do vírus da cinomose canina por RT-PCR a partir de um cão adulto com encefalomielite multifocal

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    Alexandre Mendes Amude

    2007-06-01

    Full Text Available A case of multifocal distemper encephalomyelitis in a mature dog is described. In the presented case the ante mortem clinical diagnosis of canine distemper virus (CDV infection could not be ideally performed due to the absence of typical signs of distemper, such as myoclonus and systemic signs accompanying the nervous signs. The definitive diagnosis of distemper encephalomyelitis was only carried out at post mortem through virus isolation in cell culture from fresh central nervous system (CNS fragments and CDV nucleoprotein gene detection in the CNS by RT-PCR.Descreve-se um caso de encefalomielite multifocal pela cinomose em um cão adulto. No caso apresentado o diagnóstico clínico da infecção pelo vírus da cinomose canina (CDV não pode ser adequadamente realizado devido à ausência de sinais típicos da enfermidade, tais como mioclonia e sinais sistêmicos. O diagnóstico definitivo somente foi possível post mortem pelo isolamento do CDV em cultivo celular a partir dos fragmentos frescos do sistema nervoso central (SNC e pela detecção do gene da nucleoproteína do CDV em fragmentos do SNC por meio da RT-PCR.

  12. Caracterización por PCR- múltiple del grupo filogenético de Escherichia coli uropatógena aisladas de pacientes ambulatorios de Bucaramanga, Santander

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    Alexandra Serrano

    2016-06-01

    Full Text Available Introducción: Las infecciones del tracto urinario son consideradas actualmente como una de las enfermedades infecciosas más comunes. En general se acepta que los agentes infecciosos con los cuales se asocia se encuentran presentes en la microbiota intestinal, por tal razón se ha descrito que Escherichia coli es una de las principales Enterobacterias involucradas en las manifestaciones clínicas presentes en pacientes con infecciones del tracto urinario. Las cepas de E. coli han sido clasificadas inicialmente en 4 grupos filogenéticos según Clermont y col. (2000, de este modo se conocen “cepas intestinales comensales, pertenecientes a los grupos A y B1, y cepas extra intestinales virulentas asociadas a los grupos B2 y D, para llevar a cabo dicha clasificación Clermont, diseñó herramientas moleculares como la PCR-triple, mediante la utilización de diferentes genes tales como ChuA, yjaA y TSPE4.C2”. Sin embargo debido a la complejidad estructural genética de este microorganismo, se han logrado establecer nuevos grupos filogenéticos de E. coli, A, B1, B2, C, D, E, F y un último grupo el cual no ha sido completamente identificado, por tal razón es importante conocer cuáles son los grupos filogenéticos que están presentes en nuestra región, mediante el uso de técnicas moleculares que permitan realizar la caracterización filogenética de dicho microorganismo y de esta manera lograr establecer los perfiles de resistencia y evaluar los posibles factores de virulencia asociados al proceso de infección. Objetivo: Realizar la caracterización molecular de los grupos filogenéticos de E. coli uropatógena aislada de pacientes ambulatorios que asisten a un laboratorio de tercer nivel de complejidad de la ciudad de Bucaramanga mediante el uso de PCR-multiplex. Materiales y métodos: El cálculo del tamaño muestral se realizó teniendo en cuenta la prevalencia reportada por Orduz, K y Trejos, J, (2010, se calculó el tamaño de muestra

  13. Detección de virus influenza A, B y subtipos A (H1N1 pdm09, A (H3N2 por múltiple RT-PCR en muestras clínicas

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    Pool Marcos

    Full Text Available Objetivos. Estandarizar la técnica de reacción en cadena de la polimerasa en tiempo real (RT-PCR múltiple para la detección de virus influenza A, B y tipificación de subtipos A (H1N1 pdm09, A (H3N2 en muestras clínicas. Materiales y métodos. Se analizaron 300 muestras de hisopado nasofaríngeo. Esta metodología fue estandarizada en dos pasos: la primera reacción detectó el gen de la matriz del virus de influenza A, gen de la nucleoproteína del virus influenza B y el gen GAPDH de las células huésped. La segunda reacción detectó el gen de la hemaglutinina de los subtipos A (H1N1 pandémico (pdm09 y A (H3N2. Resultados. Se identificaron 109 muestras positivas a influenza A y B, de las cuales 72 fueron positivas a influenza A (36 positivas a influenza A (H1N1 pdm09 y 36 positivos a influenza A (H3N2 y 37 muestras positivas a influenza B. 191 fueron negativas a ambos virus mediante RT-PCR en tiempo real multiplex. Se encontró una sensibilidad y especificidad del 100% al analizar los resultados de ambas reacciones. El límite de detección viral fue del rango de 7 a 9 copias/µL por virus. Los resultados no mostraron ninguna reacción cruzada con otros virus tales como adenovirus, virus sincitial respiratorio, parainfluenza (1,2 y 3, metapneumovirus, subtipos A (H1N1 estacional, A (H5N2 y VIH. Conclusiones. La RT-PCR múltiple demostró ser una prueba muy sensible y específica para la detección de virus influenza A, B y subtipos A (H1N1, H3N2 y su uso puede ser conveniente en brotes estacionales.

  14. Expressão dos genes nodC, nodW e nopP em Bradyrhizobium japonicum estirpe CPAC 15 avaliada por RT-qPCR Expression of nodC, nodW and nopP genes in Bradyrhizobium japonicum CPAC 15 strain evaluated by RT-qPCR

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    Simone Bortolan

    2009-11-01

    Full Text Available O objetivo deste trabalho foi avaliar a expressão, por RT-qPCR, dos genes de nodulação nodC e nodW e do gene nopP da estirpe CPAC 15, que provavelmente atuam na infecção das raízes da soja. Foram realizados dois experimentos. No primeiro, a expressão dos genes foi avaliada nas células após a incubação com genisteína por 15 min, 1, 4 e 8 horas. Os resultados revelaram que os três genes apresentaram maior expressão imediatamente após o contato com o indutor (15 min. No segundo experimento, a bactéria foi cultivada na presença de indutores (genisteína ou exsudatos de sementes de soja por 48 horas. A expressão dos três genes foi maior na presença de genisteína, com valores de expressão para nodC, nodW e nopP superiores ao controle. Os resultados obtidos confirmam a funcionalidade dos três genes na estirpe CPAC 15, com ênfase para o nopP, cuja funcionalidade em Bradyrhizobium japonicum foi descrita pela primeira vez.The objective of this work was to evaluate, by RT-qPCR, the expression of the nodC and nodW nodulation genes and of the nopP gene of the CPAC 15 strain, which probably play a role in the infection of soybean roots. Two experiments were done. In the first, the gene expression was evaluated in cells after incubation with genistein for 15 min, 1, 4 and 8 hours. Results showed that the three genes showed higher expression immediately after contact with the inducer (15 min. In the second experiment, the bacterium was grown in the presence of inducers (genistein or soybean seed exudates for 48 hours. The expression of the three genes was greater when induced by genistein, and the expression of nodC, nodW and nopP had higher values than the control. The results confirm the functionality of the three genes in the CPAC 15 strain, with an emphasis on the nopP, whose functionality in Bradyrhizobium japonicum was described for the first time.

  15. Avaliação laboratorial da doença residual mínima na leucemia mielóide crônica por Real-Time PCR Evaluation diagnosis of minimal residual disease in chronic myeloid leukemia by Real-Time PCR

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    Allyne Cristina Grando

    2008-12-01

    Full Text Available A leucemia mielóide crônica (LMC representa 15% das leucemias e apresenta três fases: crônica, acelerada e crise blástica. A partir da análise citogenética, pode ser identificado o cromossomo Philadelphia, característico da LMC. O transplante de células-tronco é o único tratamento curativo, mas é acompanhado de altas taxas de morbimortalidade, dificultando sua aplicação. A doença residual mínima é de grande importância para avaliar a resposta ao tratamento, tanto na verificação de doença residual, quanto na identificação de pacientes com alto risco de recaída. Muitas técnicas específicas têm sido introduzidas para detectar as translocações ou os produtos do cromossomo Philadelphia. A mais sensível é a Real-Time PCR, que detecta uma célula leucêmica em 10(5 células normais. O objetivo deste trabalho foi realizar uma revisão bibliográfica sobre a LMC, dando ênfase à utilização da técnica por Real-Time PCR.Chronic myeloid leukemia (CML represents about 15% of all leukemias and has three phases: the chronic phase, accelerated phase and blast crisis. After cytogenetic analysis, the Philadelphia chromosome, characteristic of CML, can be identificated. Stem cell transplantation is the only curative treatment for CML, but it is accompanied by high levels of morbimortality, difficulting its application. The minimal residual disease is very important for the evaluation of the response to treatment, to verify the residual disease and also to identify patients with a high risk of relapse. Many specific techniques have been introduced for the detection of translocations or products of the Philadelphia chromosome; the most sensitive being Real-Time PCR which detects 1 leukemia cell in 10(5 normal cells. The aim of this study was to perform a bibliographic review of CML, with emphasis on the utilization of the Real-Time PCR technique.

  16. Rapid detection of bovine coronavirus by a semi-nested RT-PCR Detecção rápida do Coronavírus Bovino (BCoV por meio de uma semi-nested RT-PCR

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    Karen M. Asano

    2009-11-01

    ça respiratória em bezerros. O presente estudo teve por objetivo desenvolver uma semi-nested RT-PCR para a detecção do BCoV com base em seqüências representativas e recentes do gene do nucleocapsídeo, região conservada do genoma dos coronavírus. Três primers foram desenhados, a primeira amplificação com um fragmento esperado de 463pb e a segunda (semi-nested com um fragmento esperado de 306pb. A sensibilidade analítica foi determinada pela diluição do BCoV cepa Kakegawa (título HA: 256 na base de 10 em água ultra-pura tratada com DEPC, em soro fetal bovino (SFB e em uma suspensão fecal negativa para o BCoV, onde foram encontrados resultados positivos até a diluição de 10-2, 10-3 e 10-7, respectivamente. Este resultado sugere que a quantidade total de RNA na amostra influencia na precipitação dos pellets pelo método de extração utilizado. Quando se utiliza amostra fecal, a grande quantidade de RNA total funciona como carreadora do RNA do BCoV, demonstrando elevada sensibilidade analítica e ausência de possíveis substâncias inibidoras da PCR. O protocolo final da semi-nested RT-PCR foi aplicado a 25 amostras fecais de vacas adultas, previamente avaliadas por uma nested RT-PCR RdRp utilizada como teste de referência, resultando em 20 e 17 amostras positivas para o primeiro e segundo teste, respectivamente. Os resultados dos dois sistema de diagnóstico apresentaram concordância substancial (kappa: 0,694. A elevada sensibilidade e especificidade do novo método proposto e o fato de que os primers foram desenhados baseados em sequências atuais do BCoV, oferecem bases para o diagnóstico mais acurado de infecções causadas pelo BCoV, assim como para novas perspectivas em protocolos de detecção de outros Coronavírus de importância tanto em saninade animal quanto em saúde pública.

  17. Inmunohistoquímica de la proteína p16INK4a en biopsias y extendidos cervicovaginales y su relación con HPV por PCR Immunohistochemistry of p16INK4a in biopsies and cervicovaginal smears, and its correlation with HPV detected by PCR

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    Alejandro García

    2008-12-01

    Full Text Available Estudios recientes sugieren que la sobreexpresión de p16, determinada por inmunohistoquímica, sería un marcador específico de células escamosas displásicas y neoplásicas con alta asociación con HPV de alto riesgo. Nuestro objetivo fue correlacionar los hallazgos cito/histológicos con la expresión de p16 y el subtipo de HPV por PCR. Seleccionamos 95 biopsias de cuello uterino y 4 legrados endocervicales de 99 individuos, y 30 extendidos cervicovaginales de otros 30 individuos, que se dividieron según el diagnóstico morfológico. Inmunomarcamos cortes del material incluido en parafina y los extendidos con el kit CINtecT p16INK4a (DAKO. Evaluamos HPV por PCR utilizando 25/99 biopsias con lesión intraepitelial escamosa de bajo grado. Observamos marcación positiva para p16 en 1/35 biopsias (2.9% y 1/11 extendidos (9% en los grupos sin HPV ni displasia; 16/25 biopsias (64% y 6/10 extendidos (60% en aquellos con lesión de bajo grado y 38/39 biopsias (97.4% y 8/9 extendidos (89% en los grupos con lesión de alto grado y carcinoma escamoso. Todas las muestras con HPV-6/11 fueron negativas o positivas focales para p16, en tanto que aquellas con HPV-18 u otros subtipos fueron mayoritariamente positivas de tipo difuso. Concluimos que la expresión de p16 presenta alta correlación con el diagnóstico cito/histológico y alta asociación entre la marcación difusa y la presencia de HPV de alto riesgo, aportando mayor objetividad en casos dudosos y ayudando a seleccionar grupos de individuos con riesgo de progresión de enfermedad, con un costo aceptable para estudiar grandes grupos.Recent studies suggest that p16 overexpression determined by immunohistochemistry would be a specific marker for neoplastic and dysplastic squamous cells associated with high-risk HPV. The purpose of this study was to assess the correlation between cyto-histological findings, p16 expression and HPV subtype. A total of 99 biopsies were selected, 4 endocervical

  18. Consanguinidad por isonimia en Salta

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    Albeza, María V.

    2007-01-01

    Full Text Available Se estimó el coeficiente de parentesco por isonimia para localidades de la Puna, Valle Calchaquí y Valle de Lerma, a fin de evaluar diferentes factores evolutivos que podrían estar afectando la composición genética de la población. A partir de los apellidos de las parejas consignadas en fuentes primarias de información, se estimó la isonimia conyugal o marital, el coeficiente total Ft y sus componentes Fr (inbreeding azaroso y Fn (inbreeding no azaroso. De las localidades estudiadas, en la Puna se ha detectado sólo una pareja isónima en una de ellas, en el Valle Calchaquí, tres y ninguna en el Valle de Lerma. Tanto en el Valle Calchaquí como en el de Lerma, se han estimado valores negativos de Ft, y en la Puna se registran los valores más elevados. En las localidades estudiadas no se cumple el supuesto de transmisión patrilineal de apellidos por lo que los valores de Fr y por ende de Ft podrían estar subestimados. Es por ello que sería necesario contar con información desde otras vertientes metodológicas para corroborar, complementar y manejar cuidadosamente el análisis de los datos y las conclusiones que se obtienen.

  19. Validación de la PCR en la detección de parásitos de Leishmania (Viannia spp. en Lutzomyia (Diptera: Psychodidae como herramienta en la definición de especies vectores.

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    Erika Santamaría

    2005-06-01

    Full Text Available lntroducción. En leishmaniasis se acepta que la reacción en cadena de la polimerasa (PCR ha simplificado el proceso de incriminación vectorial. Sin embargo, pocas veces se ha determinado la sensibilidad y la especificidad de cada PCR en la detección y la identificación del parasito en los flebótomos. Objetivo. Evaluar la aplicabilidad de la reacción en cadena de la polimerasa (PCR, basada en los iniciadores 81 y 82, en la detecci6n e identificación de parasitos de Leishmania (Viannia en insectos vectores enteros sin disecar. Metodología. Se determinó la sensibilidad y la especificidad de la PCR empleando diluciones de cultivo de parasitos de Leishmania spp. Se estableció el número máximo de hembras de Lutzomyia que pueden ser procesadas a la vez sin disminuir la sensibilidad de la PCR, procesando el ADN de grupos de una a cinco hembras de Lutzomyia en presencia del ADN de las diluciones de cultivo de parasitos. Además, se comparó en grupos de flebótomos infectados experimentalmente, la sensibilidad de esta PCR en la detección de infección por Leishmania (Viannia frente al método de busqueda de flagelados por disección del insecto y examen microscópico. Resultados. La PCR detectó desde un parasito de Leishmania (Viannia y permiti6 el procesamiento de hasta tres insectos enteros sin alterar la sensibilidad. Los porcentajes de infección experimental detectados con las dos tecnicas fueron similares, 33,3% con la PCR y 30% con el examen microscópico. Además, se confirmó que los iniciadores 81 y 82 son especificos para especies del subgenero Leishmania (Viannia. Conclusión. Los resultados obtenidos demuestran la sensibilidad y la especificidad de esta PCR y permiten recomendar su uso en la determinaci6n de infección natural con parasitos de Leishmania (Viannia en poblaciones silvestres de flebótomos.

  20. CARACTERIZAÇÃO MICROBIOLÓGICA POR METODOLOGIA CLÁSSICA DE DOCE DE LEITE, LEITE CONDENSADO E QUEIJO MINAS PADRÃO ADQUIRIDOS NO MERCADO DE JUIZ DE FORA (MG E PADRONIZAÇÃO DE MULTIPLEX PARA DETECÇÃO DE PATÓGENOS POR PCR EM TEMPO REAL

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    Jaqueline Flaviana Oliveira de Sá

    2012-10-01

    Full Text Available Os derivados lácteos são alimentos com excepcional valor nutritivo e amplamente consumido pela população mundial. Entretanto, são também excelentes meios de cultura para muitos micro-organismos, sendo, portanto, passíveis de contaminação por diferentes agentes microbiológicos, podendo levar a doenças manifestadas por ação de patógenos ou por suas toxinas. A obtenção de alimentos seguros depende dentre outros fatores, dos métodos de análises utilizados, os quais devem fornecer resultados rápidos e confiáveis que permitam o monitoramento da segurança microbiológica de alimentos, seja pela indústria ou pelos órgãos de fiscalização e para isso, diversos métodos alternativos têm sido desenvolvidos para a detecção e quantificação de patógenos. O primeiro objetivo do presente estudo foi caracterizar microbiologicamente, por metodologia clássica, amostras de doce de leite, leite condensado e queijo Minas Padrão com SIF, produzidos em vários estados do Brasil ecomercializados em supermercados de Juiz de Fora (MG. Foram feitas análises de contagem padrão em placas de mesófilos, bolores e leveduras, coliformes a 30ºC e a 45ºC, Staphylococcus spp. coagulase positiva e negativa, além da pesquisa de Salmonella sp. e Listeria monocytogenes. Altas contagens padrão em placas de mesófilos, leveduras e Staphylococcus spp.coagulase negativa foram encontradas nos três produtos. O segundo objetivo foi desenvolver uma metodologia alternativa à clássica, que apresentasse resultados mais rápidos e de alta especificidade para a detecção dos principais patógenos contaminantes de produtos lácteos e transmissores de doenças de origem alimentar, utilizando a técnica de PCR em tempo real. Foi padronizada uma reação multiplex para detecção de Salmonella entérica var thyphimurium e Staphylococcus aureus. O presente trabalho contribuirá com a rara literatura mundial sobre a microbiota contaminante do doce de leite

  1. Diagnosis of neonatal group B Streptococcus sepsis by nested-PCR of residual urine samples Diagnóstico de sepse neonatal causada pelo estreptococo do grupo B por meio de dupla amplificação de amostras residuais de urina

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    Bruno Nicolino Cezarino

    2008-03-01

    four patients. Moreover, PCR has enabled us to use residue volumes of urine samples collected by non invasive, non sterile methods, what is technically adequate as GBS is not part of the normal urine flora, thus avoiding invasive procedures such as suprapubic bladder punction or transurethral catheterization. At the same time, the use of urine instead of blood samples could help preventing newborns blood spoliation.O estreptococo do grupo B (GBS constitui a causa mais freqüente de sepse neonatal precoce. O teste de referência continua sendo o isolamento em cultura, apesar de apresentar problemas de sensibilidade. O objetivo do presente estudo foi validar uma técnica de dupla amplificação e determinar a possibilidade do uso de amostras residuais de urina colhidas por método não invasivo, não estéril, para a confirmação da sepse por GBS em recém-nascidos. As amostras foram amplificadas com primers do principal gene de superfície do GBS. A insuficiência de volume de material biológico para a realização de exames para suporte de vida, além de outros necessários à identificação do agente etiológico de infecções é muito freqüente em recém-nascidos. Mesmo assim, decidimos definir critérios bastante rigorosos para a inclusão de pacientes na casuística: os recém-nascidos deveriam apresentar sinais e sintomas compatíveis com infecção pelo GBS; deveriam ter tido ao menos uma amostra enviada para cultura, podendo ser sangue, urina ou líquor; disponibilidade de volumes residuais dessas amostras, ou de outras colhidas no dia da hospitalização, antes da introdução da antibioticoterapia, de forma a possibilitar a análise por PCR, e evolução favorável com a antibioticoterapia empírica. Em apenas um dos quatro recém-nascidos a infecção foi confirmada por cultura, enquanto nos outros três casos a infecção foi considerada presuntiva (pacientes preencheram os critérios de inclusão, mas o GBS não foi isolado. De um total de 12 amostras

  2. A retrospective PCR investigation of avian Orthoreovirus, chicken infectious anemia and fowl Aviadenovirus genomes contamination in commercial poultry vaccines in Brazil

    Directory of Open Access Journals (Sweden)

    P.R. Barrios

    2012-02-01

    Full Text Available Vacinas avícolas vivas comerciais produzidas entre 1991 e 2005 foram examinadas para a presença de genomas dos vírus da anemia infecciosa das galinhas (Gyrovirus CAV, da hepatite por corpúsculo de inclusão (Aviadenovirus FAdV e da artrite viral/síndrome da má absorção (Orthoreovirus aviário ARV. Vinte e seis partidas de vacinas vivas liofilizadas de oito fabricantes com lacre original foram examinadas. As extrações de DNA e PCR de CAV e FAdV, e de RNA e RT-PCR para ARV, foram descritas previamente. Contaminações triplas de ARV, CAV e FAdV foram detectadas em vacinas de mesmo fabricante, produzidas em 1991 e 1992 contra a doença de Newcastle (DN, e para a encefalomielite aviária, produzida em 1994. ARV e CAV em co-infecção foram encontrados em vacinas contra a doença de Marek liofilizadas produzidas em 1996 por dois fabricantes diferentes. Genoma de ARV foi detectado em vacinas contra a bronquite infecciosa de setembro e dezembro de 1998, doença infecciosa bursal, de dezembro de 1998 e DN de janeiro de 1998. Três dos oito fabricantes apresentaram vacinas com contaminação e cinco nunca apresentaram vacinas contaminadas. Nenhuma vacina produzida a partir de 2001 apresentou contaminação. Cogita-se um papel epidemiológico para vacinas vivas, como fonte de infecção para ARV, CAV e FAdV e, potencialmente determinante da atual alta disseminação destes.

  3. Celulitis por citomegalovirus

    Directory of Open Access Journals (Sweden)

    A. Ruiz Lascano

    2002-12-01

    Full Text Available Las lesiones cutáneas por citomegalovirus (CMV son infrecuentes y a menudo una manifestación tardía de una enfermedad sistémica, que generalmente anuncia un curso fatal. Comunicamos un caso de celulitis por CMV: una mujer de 70 años con trasplante renal efectuado 1 mes antes de la consulta, terapia inmunosupresora con ciclosporina A y metilprednisona. La paciente ingresó por fiebre, dolor e impotencia funcional en pierna derecha. Comprobamos la existencia de una placa de 8 por 4 cm eritematoedematosa. La tratamos con antibióticos sin mejoría, por lo que realizamos un estudio histopatológico de piel que mostró cambios citopáticos compatibles con infección por CMV. Los cultivos bacteriológicos y micológicos fueron negativos. La inmunohistoquímica específica para CMV y el estudio de reacción en cadena de la polimerasa (PCR de la biopsia de piel fueron positivas, al igual que la antigenemia. El tratamiento con ganciclovir produjo la mejoría del cuadro clínico. En la literatura revisada no hemos encontrado la celulitis como manifestación de enfermedad cutánea por CMV.

  4. Desarrollo y evaluación de una prueba de reacción en cadena de la polimerasa (PCR, utilizando la secuencia del gen hilA para diagnóstico de fiebre entérica por Salmonella spp.

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    Miryan Margot Sánchez

    2004-06-01

    Full Text Available El diagnóstico de fiebre entérica por Salmonella spp. se basa en el aislamiento de la bacteria en hemocultivos el cual consume tiempo, no siempre está disponible y tiene poca utilidad en pacientes con tratamiento antibiótico previo. Por consiguiente, se hace necesario el desarrollo de una prueba rápida, sensible y específica para el diagnóstico de fiebre entérica. Salmonella spp. utiliza el gen hilA (componente de la isla de patogenicidad I para invadir células epiteliales y producir infección. Al usar la secuencia de este gen se diseñó una prueba de PCR para detectar la bacteria en sangre y se evaluó su sensibilidad, especificidad, valor predictivo positivo y valor predictivo negativo, utilizando la metodología prueba de una prueba. La prueba de oro fue el hemocultivo. Se estudiaron 34 individuos con sintomatología de fiebre entérica con aislamiento de Salmonella serotipo Typhi en hemocultivos; 35 individuos con sepsis por otros bacilos Gram negativos aislados de hemocultivo (Klebsiella pneumoniae, 9; Serratia marcescens, 5; Escherichia coli, 4; Pseudomonas aeruginosa, 9; Providencia alcalifaciens, 4, y Enterobacter cloacae, 4 y 150 muestras de sangre de voluntarios asintomáticos. La sensibilidad, especificidad, valor pronóstico positivo y valor pronóstico negativo de la PCR fue del 100%. El número mínimo de UFC/ml que la PCR detecta en sangre es de 10.

  5. Rapid diagnosis and identification by PCR of Yersinia ruckeri isolated of Oncorhynchus mykiss from Canta, Lima, Peru Diagnóstico e identificación rápidos por PCR de Yersinia ruckeri aislada de Oncorhynchus mykiss procedentes de Canta, Lima, Perú

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    Susana Sirvas-Cornejo

    2012-02-01

    Full Text Available Twenty individuals of rainbow trout were sampled (fry and juveniles from Acochinchan Fishfarm (Canta, Lima - Peru, and analyzed with the Polimerase Chain Reaction test (PCR in order to achieve a rapid identification of Yersinia ruckeri, which is the pathogen agent that causes the enteric red mouth disease (ERM and produces high rates of mortality. Nine fish samples were asymptomatic, while 11 of them showed signs of ERM. In addition, 22 bacterial strains were isolated from the liver, spleen and kidney. PCR and specific primers (16S rRNA, were used to amplified a specific 575 bp DNA fragment of Yersinia ruckeri. Nineteen strains were identified as Yersinia ruckeri by PCR in symptomatic and asymptomatic fishes. It was established a diagnosis time of 26 hours, compared with the 2 or 3 days that would take the diagnosis using biochemical tests.Se muestrearon 20 ejemplares (alevines y juveniles de trucha arco iris cultivados en la piscifactoría Acochinchán (Canta, Lima, Perú, y se les aplico la técnica de la Reacción en Cadena de la Polimerasa (PCR con la finalidad de obtener una identificación rápida del agente patógeno Yersinia ruckeri que produce la enfermedad entérica de la boca roja (ERM y genera elevadas tasas de mortalidad. Nueve ejemplares fueron asintomáticos mientras que 11 presentaron signos de ERM. Se aislaron 22 cepas bacterianas del hígado, bazo y riñón. Se empleó la técnica de la PCR para la amplificación y cebadores específicos (ARNr 16S, que permitieron amplificar un fragmento de ADN de 575 pb de Yersinia ruckeri. Diecinueve cepas fueron identificadas como Yersinia ruckeri mediante la PCR, tanto en peces sintomáticos como asintomáticos. Se estableció un tiempo de diagnóstico de 26 horas, en comparación con los 2 ó 3 días que duraría el diagnóstico empleando las pruebas bioquímicas.

  6. Detection of poliovirus type 2 in oysters by using cell culture and RT-PCR Detecção de poliovírus tipo 2 em ostras através de cultura celular e RT-PCR

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    Cecília E.B. Vinatea

    2006-03-01

    ância em saúde pública. No presente estudo, ostras foram colocadas em aquários de vidro contendo água do mar adicionada de algas unicelulares. Dois tipos de experimentos foram realizados: a ostras bioacumulando quatro diferentes concentrações de poliovírus: 5 x 10(4, 2,5 x 10(4, 5 x 10³, 5 x 10² PFU/mL durante 20h; b tecidos de ostras inoculados diretamente com 6,0 x 10(5 e 1,0 x 10(5 PFU/mL. Após a semeadura, os tecidos foram processados por um método de adsorção-eluição-precipitação. Controles positivos foram realizados por inoculação de 6,0 x 10(5 PFU/mL de poliovírus diretamente nos tecidos processados das ostras. Os extratos teciduais foram testados para presença do vírus por ensaios de placa de lise (PFU, RT-PCR e cultura celular integrada ao PCR (ICC/PCR. Este último consistiu na inoculação das amostras sobre monocamadas de células VERO seguida de RT-PCR do fluido celular infeccioso. No primeiro experimento (ensaio de bioacumulação por 20h, foram detectados até 5 x 10³ PFU de poliovírus, após 24 e 48h de replicação nas células. Os ensaios de RT-PCR e ICC/PCR foram capazes de detectar 3 e 0,04 PFU de poliovírus, respectivamente nos ensaios de bioacumulação. Quando os extratos teciduais processados foram semeados, os ensaios de placa de lise demonstraram recuperação de vírus infecciosos em todas as concentrações testadas. Pudemos concluir que partículas viáveis de poliovírus podem ser detectadas em ostras após bioacumulação e que estas técnicas podem ser diretamente aplicadas na detecção de vírus em amostras ambientais.

  7. Aplicación de la técnica de PCR en la detección de Ralstonia solanacearum en campos paperos

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    Analía Sanabria

    2013-01-01

    Full Text Available La murchera de la papa causada por Ralstonia solanacearum es una amenaza permanente para el cultivo con consecuencias devastadoras en condiciones predisponentes. La incidencia de esta enfermedad en Uruguay es variable; desde la década de 1970 se registran brotes agudos periódicos difíciles de prevenir y controlar. Se ha detectado a R. solanacearumen todas las regiones de producción, lo que dificulta el mantenimiento de áreas libres de patógeno y la continuidad del cultivo. Dado que no existen agentes químicos efectivos, las estrategias de control apuntan a la prevención, recomendando la rotación de cultivos, la desinfección de herramientas y el uso de semilla certificada libre del patógeno. En este trabajo se optimizó la detección molecular de R. solanacearum en suelo mediante PCR con el objetivo de conocer la persistencia del patógeno en el campo. El método se aplicó al análisis de tres campos con diferentes antecedentes de murchera, y se demostró que el patógeno es capaz de permanecer viable por más de 10 años. También se determinaron las ventajas y limitaciones de esta metodología en su aplicación a muestras complejas. Estos resultados constituyen un importante aporte que genera conocimiento que permitirá a los productores delinear mejores estrategias para el manejo de la enfermedad al momento de planificar los cultivos.

  8. CORRELACIÓN ENTRE LAS LESIONES MACROSCÓPICAS E HISTOPATOLÓGICAS DE LA NEUMONÍA ENZOÓTICA Y LA DETECCIÓN DEL Mycoplasma hyopneumoniaePOR PCR ANIDADA EN LAVADOS BRONCO ALVEOLARES EN CERDOS AL SACRIFICIO

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    H Guzmán

    2008-01-01

    Full Text Available Mycoplasma hyopneumoniae es el agente etiológico primario de la neumonía enzoótica (ne de los porcinos, y es el agente de mayor importancia involucrado en el Complejo res-piratorio Porcino (CrP. el propósito de este trabajo fue evaluar las lesiones en pulmones de 55 cerdos en planta de sacrificio procedentes de granjas de producción intensiva; las muestras fueron tomadas en forma aleatoria con base en lesiones sugestivas de ne y pulmo-nes aparentemente normales como controles para análisis histopatológico y para detección de adn de Mycoplasma hyopneumoniae en lavados bronco alveolares por PCr anidada. Las lesiones macroscópicas fueron evaluadas en términos de porcentaje de afección y las lesiones histopatológicas fueron clasificadas (de 0-4 según escala de severidad subjetiva, de acuerdo con el grado de hiperplasia de agregados linfoides asociados a bronquios, bron-quiolos y vasos sanguíneos (BaLT. Mediante la técnica de PCr anidada fueron positivas 54 de 55 muestras. Las lesiones histopatológicas del BaLT mostraron alta correlación con los hallazgos macroscópicos y con lesiones microscópicas de hiperplasia de células epite-liales e infiltración de células inflamatorias en vías aéreas. Los resultados demostraron que el PCr anidado es una herramienta complementaria importante para el diagnóstico de la presencia de Mycoplasma hyopneumoniae en afecciones respiratorias asociadas con ne y CRP de los porcinos al sacrificio.

  9. Detección de anticuerpos antiplasmodium por ELISA en donantes de sangre

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    Patricia Olaya de Morales

    1982-06-01

    Full Text Available La malaria, una enfermedad transmitida por mosquitos del genero anopheles, puede ser inducida a través de transfusiones de sangre infectada con alguna de las especies de Plasmodium que afectan al hombre. Con el objeto de determinar el riesgo potencial de infección inducida por transfusiones, se analizaron durante 9 meses y mediante la técnica de E.L.I.S.A., las muestras de suero tomadas a los donantes de sangre del Hospital Militar Central de Bogotá. El 8.6 por mil de las 3114 muestras analizadas, resultaron positivas para anticuerpos antimaláricos y durante el tiempo del estudio fueron detectados 3 casos de malaria inducida por transfusiones.

  10. Teores de nutrientes minerais e metais pesados em açúcar mascavo produzido por diferentes sistemas orgânicos e convencionais

    OpenAIRE

    Paulo Dirceu Luchini

    2014-01-01

    Quantificou-se os teores de Cu, Zn, Mn, Fe, Pb e Cd em açúcar mascavo oriundo de canas produzidas por diferentes formas de cultivo de sistemas orgânicos e convencionais. Os metais Pb e Cd foram determinados por espectrometria de absorção atômica com atomização eletrotérmica em forno de grafite (GFAA) e os metais Cu, Zn, Mn e Fe foram determinados por espectrometria de absorção atômica com chama (FAAS). Os teores de Cu, Zn, Mn e Fe foram detectados apenas em valores abaixo do limite recomendad...

  11. Eliminating PCR contamination

    International Nuclear Information System (INIS)

    Fox, J.C.; Ait-Khaled, Mounir; Webster, Alison; Emery, V.C.

    1991-01-01

    The sensitivity of polymerase chain reaction (PCR) can mean that even very low levels of contamination with the target DNA will result in a positive signal. At present this aspect is a major limitation in the use of PCR as a routine diagnostic method. By exposing PCR reagents to UV light, contaminating DNA can be inactivated, thus providing an opportunity to eradicate false positive reactions. UV irradiation was applied to PCR systems used for detection of human cytomegalovirus CMV and human immunodeficiency virus (HIV) and shown to be effective in eradicating both laboratory encountered contamination and plasmid DNA (below 100 pg) added to PCR systems prior to UV exposure. Sensitivity of a PCR system to amplify the long terminal repeat (LTR) sequence of HIV-1 was not affected by the irradiation procedure; however, ultimate sensitivity of a PCR system for the amplification of an early gene pro-motor sequence of the CMV genome was reduced 1000-fold. UV irradiation did not affect the size of the PCR product as determined by strand separating polyacrylamide gel electrophoresis of a 32 P-labelled amplimer. Thus, a simple pre-exposure to UV light would seem a worth-wile step to incorporate into PCR protocols provided that the effects on sensitivity have been determined empirically for each PCR system. (author). 11 refs.; 3 figs

  12. Identificação do circovírus suíno tipo 2 por reação em cadeia da polimerase e por imunoistoquímica em tecidos suínos arquivados desde 1988 no Brasil

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    Ciacci-Zanella Janice Reis

    2006-01-01

    Full Text Available A síndrome multissistêmica do definhamento dos suínos (SMDS é uma doença de importância econômica causada pelo circovírus suíno tipo 2 (PCV2. Uma pesquisa retrospectiva foi realizada em amostras de órgãos de suínos fixados em blocos de parafina arquivados, que haviam sido submetidos à Embrapa Suínos e Aves entre 1985 e 1998 para o diagnóstico histopatológico. Vinte e cinco casos foram selecionados com base nas lesões histológicas características da SMDS, tais como linfoadenopatia, pneumonia intersticial, hepatite e nefrite intersticial. A presença de PCV2 nos cortes histológicos foi pesquisada por reação em cadeia da polimerase interna (nested-PCR, na qual utilizou-se primers específicos para a seqüência da ORF2 do PCV2 e também por imunoistoquímica, utilizando um anticorpo monoclonal específico para o capsídeo do PCV2. O DNA viral e os antígenos específicos do PCV2 foram detectados em amostras de tecidos de dois dos 25 casos analisados, sendo um desses datado de 1988. Esses resultados indicam que o PCV2 já estava presente no Brasil desde 1988.

  13. Correlación entre la tipificación capsular de aislamientos colombianos de Haemophilus influenzae por el método de aglutinación en lámina y la técnica de PCR.

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    Marylin Hidalgo

    2003-06-01

    Full Text Available En 1998 se inició en Colombia la inmunización de niños menores de un año de edad con la vacuna conjugada contra Haemophilus influenzae, serotipo b. En el 2000, el programa de vigilancia del Grupo de Microbiología del Instituto Nacional de Salud informó una disminución de 40% de los casos de meningitis por este microorganismo, la cual se atribuyó a la vacunación. En este programa de vigilancia se utiliza de rutina la técnica estandarizada de aglutinación en lámina para la tipificación capsular de H. influenzae. El objetivo de este trabajo fue establecer la concordancia entre la prueba de aglutinación en lámina y la técnica de PCR. Se estudiaron con ambas técnicas 146 aislamientos clínicos invasores de H. influenzae, obtenidos de niños menores de 5 años, recolectados a partir de 1999 hasta 2002, identificados y serotipificados por el laboratorio de referencia como parte de la vigilancia de la meningitis bacteriana aguda y la infección respiratoria aguda. Nuestros resultados mostraron una correlación de 93% en la tipificación capsular de H. influenzae, serotipo b, y de 92% con respecto al resto de serotipos. La técnica de aglutinación en lámina realizada con un estricto control de calidad continúa siendo una herramienta sensible y específica para la serotipificación de H. influenzae.

  14. External PCR, ASN's decision

    International Nuclear Information System (INIS)

    Anon.

    2012-01-01

    The French law imposes in some situations the presence of a person skilled in radiation protection (PCR). This article describes the cases when this person must belong to the staff of the enterprise or when this person may be sub-contracted. For instance in most nuclear facilities the PCR must be on the payroll, for enterprises dedicated to nuclear transport the PCR's job can be sub-contracted. A decision given by the ASN (French Nuclear Safety Authority) sets the minimal requests (in terms of training, job contract, activities) of the sub-contracted PCR. (A.C.)

  15. Inverse fusion PCR cloning.

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    Markus Spiliotis

    Full Text Available Inverse fusion PCR cloning (IFPC is an easy, PCR based three-step cloning method that allows the seamless and directional insertion of PCR products into virtually all plasmids, this with a free choice of the insertion site. The PCR-derived inserts contain a vector-complementary 5'-end that allows a fusion with the vector by an overlap extension PCR, and the resulting amplified insert-vector fusions are then circularized by ligation prior transformation. A minimal amount of starting material is needed and experimental steps are reduced. Untreated circular plasmid, or alternatively bacteria containing the plasmid, can be used as templates for the insertion, and clean-up of the insert fragment is not urgently required. The whole cloning procedure can be performed within a minimal hands-on time and results in the generation of hundreds to ten-thousands of positive colonies, with a minimal background.

  16. Molecular diagnostic PCR handbook

    International Nuclear Information System (INIS)

    Viljoen, G.J.; Crowther, J.R.; Nel, L.H.

    2005-01-01

    The uses of nucleic acid-directed methods have increased significantly in the past five years and have made important contributions to disease control country programmes for improving national and international trade. These developments include the more routine use of PCR as a diagnostic tool in veterinary diagnostic laboratories. However, there are many problems associated with the transfer and particularly, the application of this technology. These include lack of consideration of: the establishment of quality-assured procedures, the required set-up of the laboratory and the proper training of staff. This can lead to a situation where results are not assured. This book gives a comprehensive account of the practical aspects of PCR and strong consideration is given to ensure its optimal use in a laboratory environment. This includes the setting-up of a PCR laboratory; Good Laboratory Practice and standardised PCR protocols to detect animal disease pathogens. Examples of Standard Operating Procedures as used in individual specialist laboratories and an outline of training materials necessary for PCR technology transfer are presented. The difficulties, advantages and disadvantages in PCR applications are explained and placed in context with other test systems. Emphasis is placed on the use of PCR for detection of pathogens, with a particular focus on diagnosticians and scientists from the developing world. It is hoped that this book will enable readers from various disciplines and levels of expertise to better judge the merits of PCR and to increase their skills and knowledge in order to assist in a more logical, efficient and assured use of this technology

  17. Síndrome pulmonar por hantavirus en población infantil. Chile: Regiones IX y X. 1998-2000

    OpenAIRE

    SOZA C., GUILLERMO; LORCA O., PEDRO; PUEBLA M., SERGIO; WENZEL M., MARISOL; NAVARRETE C., MARITZA; VILLAGRA C., ELIECER; MORA R., JUDITH; LEVIS C., SILVANA; AVILES A., GABRIELA

    2000-01-01

    El síndrome pulmonar por hantavirus (SPH) ha estado presente en Chile desde 1993 y ha sido detectado desde 1997 en la IX Región. Es una grave zoonosis con alta mortalidad, que afecta a gente joven incluyendo niños. Se ha estimado oportuno dar a conocer nuestra experiencia en la atención de 6 pacientes pediátricos, atendidos en las unidades de Cuidados Intensivos y Aislamiento en el Hospital Regional de Temuco, entre enero de 1998 y enero de 2000 mediante un estudio descriptivo de la experienc...

  18. Celulitis por citomegalovirus Cytomegalovirus cellulitis

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    A. Ruiz Lascano

    2002-12-01

    Full Text Available Las lesiones cutáneas por citomegalovirus (CMV son infrecuentes y a menudo una manifestación tardía de una enfermedad sistémica, que generalmente anuncia un curso fatal. Comunicamos un caso de celulitis por CMV: una mujer de 70 años con trasplante renal efectuado 1 mes antes de la consulta, terapia inmunosupresora con ciclosporina A y metilprednisona. La paciente ingresó por fiebre, dolor e impotencia funcional en pierna derecha. Comprobamos la existencia de una placa de 8 por 4 cm eritematoedematosa. La tratamos con antibióticos sin mejoría, por lo que realizamos un estudio histopatológico de piel que mostró cambios citopáticos compatibles con infección por CMV. Los cultivos bacteriológicos y micológicos fueron negativos. La inmunohistoquímica específica para CMV y el estudio de reacción en cadena de la polimerasa (PCR de la biopsia de piel fueron positivas, al igual que la antigenemia. El tratamiento con ganciclovir produjo la mejoría del cuadro clínico. En la literatura revisada no hemos encontrado la celulitis como manifestación de enfermedad cutánea por CMV.Cutaneous lesions in CMV infection are rare, often a late manifestation of systemic infection, and usually herald a fatal course. A 70 year-old woman received a kidney transplantation one month before consulting and immunosuppressive therapy that included cyclosporine A and methylprednisone. She complained of fever, local pain in her right leg, and an erythematous and swelling plaque. She was treated with intravenous antibiotics without improvement. A skin biopsy was performed and the tissue obtained was sent for bacterial and fungal cultures as well as for histological examination. Cultures were negative. The biopsy showed CMV cytopathic changes. Immunoperoxidase staining was positive for CMV and polymerase chain reaction (PCR testing revealed CMV DNA. She was treated with ganciclovir with resolution of the lesion. CMV cellulitis is a rare cutaneous manifestation

  19. Prevalência e localização espacial dos casos de tracoma detectados em escolares de Botucatu, São Paulo - Brasil Prevalence and spatial distribution of trachoma among schoolchildren in Botucatu, São Paulo - Brazil

    Directory of Open Access Journals (Sweden)

    Silvana Artioli Schellini

    2010-08-01

    Full Text Available OBJETIVO: Avaliar a prevalência de tracoma em escolares de Botucatu/SP-Brasil e a distribuição espacial dos casos. MÉTODOS: Foi realizado um estudo transversal, em crianças de 7-14 anos, que frequentavam as escolas do ensino fundamental de Botucatu/SP, em novembro/2005. O tamanho da amostra foi estimado em 2.092 crianças, considerando-se a prevalência histórica de 11,2%, aceitando-se erro de estimação de 10% e nível de confiança de 95%. A amostra foi probabilística, ponderada e acrescida de 20%, devido à possível ocorrência de perdas. Examinaram-se 2.692 crianças. O diagnóstico foi clínico, baseado na normatização da Organização Mundial da Saúde (OMS. Para avaliação dos dados espaciais, utilizou-se o programa CartaLinx (v1.2, sendo os setores de demanda escolar digitalizados de acordo com as divisões do planejamento da Secretaria de Educação. Os dados foram analisados estatisticamente, sendo a análise da estrutura espacial dos eventos calculadas usando o programa Geoda. RESULTADOS: A prevalência de tracoma nos escolares de Botucatu foi de 2,9%, tendo sido detectados casos de tracoma folicular. A análise exploratória espacial não permitiu rejeitar a hipótese nula de aleatoriedade (I= -0,45, p>0,05, não havendo setores de demanda significativos. A análise feita para os polígonos de Thiessen também mostrou que o padrão global foi aleatório (I= -0,07; p=0,49. Entretanto, os indicadores locais apontaram um agrupamento do tipo baixo-baixo para um polígono ao norte da área urbana. CONCLUSÃO: A prevalência de tracoma em escolares de Botucatu foi de 2,9%. A análise da distribuição espacial não revelou áreas de maior aglomeração de casos. Embora o padrão global da doença não reproduza as condições socioeconômicas da população, a prevalência mais baixa do tracoma foi encontrada em setores de menor vulnerabilidade social.PURPOSE: To assess the prevalence of trachoma in schoolchildren of Botucatu

  20. PCR in forensic genetics

    DEFF Research Database (Denmark)

    Morling, Niels

    2009-01-01

    Since the introduction in the mid-1980s of analyses of minisatellites for DNA analyses, a revolution has taken place in forensic genetics. The subsequent invention of the PCR made it possible to develop forensic genetics tools that allow both very informative routine investigations and still more...... and more advanced, special investigations in cases concerning crime, paternity, relationship, disaster victim identification etc. The present review gives an update on the use of DNA investigations in forensic genetics.......Since the introduction in the mid-1980s of analyses of minisatellites for DNA analyses, a revolution has taken place in forensic genetics. The subsequent invention of the PCR made it possible to develop forensic genetics tools that allow both very informative routine investigations and still more...

  1. Evaluation of PCR and multiplex PCR in relation to nested PCR for diagnosing Theileria equi

    Directory of Open Access Journals (Sweden)

    Danielle C. Leal

    2011-07-01

    Full Text Available Conventional PCR (PCRTeq for diagnosing Theileria equi and multiplex PCR (M/PCRTeq-Bc for diagnosing T. equi and Babesia caballi were comparatively evaluated with nested PCR (N/PCR-Teq for diagnosing equine piroplasmosis. In DNA sensitivity determinations, in multiple dilutions of equine blood that had tested positive for T. equi, PCR-Teq and N/PCR-Teq detected hemoparasite DNA in the larger dilutions (1:128, but did not differ significantly from the M/PCRTeq-Bc (1:64. In analyses on equine serum tested by ELISA, there was high agreement between this serological test and PCR-Teq (k = 0.780 and moderate agreement with N/PCR-Teq (k = 0.562 and M/PCRTeq-Bc (k = 0.488. PCR-Teq found a higher frequency of T. equi both in extensively and intensively reared horses, but this was not significant in relation to N/PCR-Teq (P>0.05, and both PCRs indicated that there was an endemic situation regarding T. equi in the population of horses of this sample. PCR-Teq was only significantly different from M/PCR-Teq-Bc (P<0.05. PCR-Teq presented high sensitivity and specificity, comparable to N/PCR-Teq, but with the advantage of higher speed in obtaining results and lower costs and risks of laboratory contamination. This accredits PCR-Teq for epidemiological studies and for determinations on affected horses.

  2. Aspectos epidemiológicos das infecções por rota vírus no Distrito Federal, Brasil

    Directory of Open Access Journals (Sweden)

    José Marcus Sócrates Teixeira

    1991-12-01

    Full Text Available Rotavírus foram pesquisados em 607amostrasfecais de crianças de até 6 anos de idade com quadros de diarréia aguda, no período de maio de 1986 a abril de 1990. Foi utilizada a técnica de eletroforese em gel depoliacrilamida (PAGE, sendo os rotavírus detectados em 123 amostras (20,27% das quais 107(87,00% apresentaram perfil eletroforético longo, compatível com o subgrupo II. Os rotavírus não foram encontrados no grupo controle constituído de crianças sadias, sendo porém detectados em 7,80% das crianças internadas por outras causas que não diarréia aguda. A maioria das crianças positivas para rotavírus encontrava-se na faixa etária de 6a24 meses (73,98%. A média depositividade nos meses chuvosos (outubro a abril foi igual a 9,60% e no período seco, 34,48% com picos que variaram entre 53,17 e 73,27% nos meses de junho e julho, os mais frios do ano.

  3. PCR, exit stage left ...

    CERN Multimedia

    2004-01-01

    The Prevessin Control Room during LEP's start up in 1989. The Prévessin Control Room (PCR) was recently engulfed in a wave of nostalgia. The PCR, scene of some of the greatest moments in CERN's history, is being dismantled to prepare for a complete overhaul. In February 2006, a new combined control centre for all the accelerators will open its doors on the same site, together with a new building currently under construction (see Bulletin issue 27/2004 of 28 June 2004). This marks the end of an important chapter in CERN's history. The Prévessin Control Room saw its first momentous event 28 years ago when the 400 GeV beam for the SPS was commissioned in the presence of Project Leader John Adams. It was also here that the first proton-antiproton collisions were observed, in 1981. Eight years later, in 1989, operators and directors alike jumped for joy at the announcement of the first electron-positron collisions at the start up of LEP, the biggest accelerator in the world. Today the 80 terminals and PCs have b...

  4. Lozas inglesas desechadas por los miembros de la administración de Alexandra Colony, 1870-1885, Santa Fe, Argentina

    OpenAIRE

    Dosztal, Irene

    2017-01-01

    Como ejemplo de un proyecto de colonización oficial, Alexandra Colony contaba con un centro administrativo que regulaba su desarrollo. Conocidas como Casas Centrales de Administración cumplieron diferentes funciones: residencial, comercial-administrativa y centro social. La identificación y estudio del conjunto cerámico detectados en dos pozos de basura ubicados en sus alrededores, es uno de los ejes que nos llevará a conocer el modo de vida cotidiano llevado por los diferentes directores y s...

  5. Reporte de insuficiencia renal producida por Leptospira Interrogans Serovar Pomona en Colombia

    Directory of Open Access Journals (Sweden)

    Fortunato Ospino

    1988-06-01

    Full Text Available Se describe la presentación de un caso de leptospirosis producida por L. pomona en un paciente de 20 años de edad, trabajadora habitual en un molino de arroz y caracterizado por un síndrome de insuficiencia renal y hepática. El cuadro clínico consistió principalmente en malestar general, anorexia, vómito, fiebre, diarrea, ictericia y hemorragias subconjuntivales. Los exámenes de laboratorio mostraron alteraciones, en los niveles de nitrógeno ureico, creatinina, depuración de creatinina, concentración de cloro, fosfatasa alcalina y bilirrubina directa lo cual podría indicar un daño en el funcionamiento hepático y renal. Las alteraciones en la velocidad de sedimentación, hemoglobina, hematocrito y leucocitos (Tabla 1 explican la anemia e infección ocasionada por la L. pomona en la paciente. El diagnóstico de leptospirosis fue confirmado por el aislamiento del microorganismo de la orina a pesar de no haber detectado anticuerpos en el suero de la paciente. Se pone en evidencia la necesidad de realizar siempre el diagnóstico diferencial con leptospirosis en todo síndrome hepatorrenal, especialmente en los casos con antecedentes epidemiológicos o que por la sintomatología clínica se sospeche la infección.

  6. Avaliação da urina e de leucócitos como amostras biológicas para a detecção ante mortem do vírus da cinomose canina por RT-PCR em cães naturalmente infectados Evaluation of the urine and leucocytes as biological samples for ante mortem detection of canine distemper virus by RT-PCR assay in naturally infected dogs

    Directory of Open Access Journals (Sweden)

    F.J. Negrão

    2007-02-01

    Full Text Available Urine and leucocytes were comparatively evaluated as clinical samples for ante mortem detection of the canine distemper virus (CDV by a reverse transcription-polymerase chain reaction (RT-PCR assay. One hundred and eighty eight dogs with clinical symptoms of distemper, were distributed in three groups. The group A was constituted of 93 dogs with systemic signs of distemper; the group B by 11 dogs with neurological signs, and the group C by 84 dogs that presented simultaneously systemic and neurological signs. In 66.5% (125/188 of the dogs was amplified an amplicon with 287 base pair of the CDV nucleoprotein gene. In 60.8% (76/125 of the animals the CDV was detected simultaneously in the urine and leucocytes, and in 39.2% (49/125 of the dogs just a type of clinical sample (urine: n=37; leucocytes: n=12 was positive. These results demonstrate that the different forms of clinical distemper disease can hinder the choice of only one type of clinical sample to carry out the ante mortem etiological diagnosis of CDV infection, and false-negative results can be generated.

  7. Quantitative (real-time) PCR

    International Nuclear Information System (INIS)

    Denman, S.E.; McSweeney, C.S.

    2005-01-01

    Many nucleic acid-based probe and PCR assays have been developed for the detection tracking of specific microbes within the rumen ecosystem. Conventional PCR assays detect PCR products at the end stage of each PCR reaction, where exponential amplification is no longer being achieved. This approach can result in different end product (amplicon) quantities being generated. In contrast, using quantitative, or real-time PCR, quantification of the amplicon is performed not at the end of the reaction, but rather during exponential amplification, where theoretically each cycle will result in a doubling of product being created. For real-time PCR, the cycle at which fluorescence is deemed to be detectable above the background during the exponential phase is termed the cycle threshold (Ct). The Ct values obtained are then used for quantitation, which will be discussed later

  8. One-stop polymerase chain reaction (PCR): An improved PCR ...

    African Journals Online (AJOL)

    Yomi

    2011-12-21

    Dec 21, 2011 ... membrane filtration was carried out with a commercial PCR product purification kit (Generay, Shanghai), according to the manufacture's instruction. In brief, 50 µl PCR product was mixed thoroughly with binding buffer, and the resultant mixture was loaded directly onto a silica membrane Gelclean column.

  9. Digital PCR: A brief history

    OpenAIRE

    Morley, Alexander A.

    2014-01-01

    Digital PCR for quantification of a target of interest has been independently developed several times, being described in 1990 and 1991 using the term “limiting dilution PCR” and in 1999 using the term “digital PCR”. It came into use in the decade following its first development but its use was cut short by the description of real-time PCR in 1996. However digital PCR has now had a renaissance due to the recent development of new instruments and chemistry which have made it a much simpler and...

  10. Caracterización clínica y molecular de individuos con el síndrome frágil X detectados por análisis inmunohistoquímico Clinical and molecular characterization of individuals with fragile X syndrome detected by immunohistochemical analysis

    Directory of Open Access Journals (Sweden)

    Iván Hernández García

    2004-09-01

    Full Text Available Se realizó el estudio de 50 varones con retraso mental y/o autismo de etiología desconocida a fin de detectar individuos afectados con el síndrome frágil X a través del análisis inmunohistoquímico y caracterizarlos clínica y molecularmente. Entre los casos pesquisados se detectaron 3 individuos con baja expresión de la proteína relacionada con el síndrome, a quienes se les realizó la caracterización molecular. La correlación inmunohistoquímica y molecular en 2 de ellos fue positiva. La no correlación de un tercero sugiere que pudiera tratarse de una mutación puntual o una deleción del gen relacionado con la enfermedad. En otro paciente con el fenotipo neuropsicológico y físico característicos de la enfermedad se observó una expresión promedio normal baja, lo que motivó la indicación de la caracterización molecular, que resultó ser positiva. Se discuten los mecanismos genéticos y fisiopatológicos que pudieran explicar la presencia de la proteína en las células analizadas.50 males with mental retardation and/or autism of unknown etiology were studied aimed at detecting individuals affected with the Fragile X syndrome by the immunohistochemical analysis and at characterizing them from the clinical and molecular point of view. Among the screneed subjects, 3 individuals with low expression of the protein related to the syndrome were detected. Molecular characterization was performed in these cases. The immunohistochemical and molecular correlation was positive in 2 of them. The non correlation of the third suggests that it may be a punctual mutation or a deletion of the gene connected with the disease. In another patient with the neuropsychological and physical phenotype characteristic of the disease, it was observed an average normal low expression that led to the indication of the molecular characterization, which proved to be positive. The genetic and physiopathological mechanisms that could explain the presence of the protein in the analyzed cells are discussed.

  11. PCR em tempo real para diagnóstico da leucose enzoótica bovina Enzootic bovine leukosis real time PCR

    Directory of Open Access Journals (Sweden)

    Natanael Lamas Dias

    2012-08-01

    Full Text Available O objetivo deste trabalho foi realizar a validação de uma reação em cadeia da polimerase em tempo real com o sistema Plexor® (qPCR para o diagnóstico da Leucose Enzoótica Bovina (LEB, por meio da comparação com testes de diagnóstico recomendados pela Organização Mundial de Saúde Animal (OIE. A qPCR foi comparada com duas outras técnicas: a PCR nested (nPCR e a imunodifusão em gel de ágar (IDGA. Das 82 amostras analisadas pela qPCR e nPCR, 79 apresentaram resultados concordantes, sendo a concordância, classificada pelo Índice Kappa, como alta. Entre as PCRs e a IDGA, o número de resultados concordantes foi de 71 e 69, respectivamente, para qPCR e nPCR, sendo a concordância classificada como considerável. A qPCR apresentou altos valores de sensibilidade e especificidade. Os valores preditivos da qPCR observados demonstraram a alta capacidade de classificação dos casos positivos e negativos. A qPCR não foi capaz de detectar três amostras positivas e tem custo ligeiramente superior que a nPCR. Entretanto, a qPCR é uma técnica mais rápida, menos susceptível a contaminações, tem alta sensibilidade, não utiliza e não gera resíduos carcinogênicos. Concluímos que a qPCR pode substituir a nPCR recomendada pela OIE no diagnóstico de rotina em áreas em que a LEB é endêmica, como no Brasil.The goal of this research was to validate a Plexor® real time Polymerase Chain Reaction (qPCR for Enzootic Bovine Leukosis (EBL diagnosis by comparison with methods recommend by the World Animal Health Organization (OIE. The qPCR was compared with two other techniques: the nested PCR (nPCR and to the agar gel immunodiffusion (AGID. Of 82 qPCR and nPCR analysed samples, 79 presented concordant results, being the concordance classified by Kappa Index as high. Between the PCRs and AGID, the number of concordant results was 71 and 69, out of 82, to qPCR and nPCR, respectively, being the concordance classified as considerable, in both

  12. Methylation-Specific PCR Unraveled

    Directory of Open Access Journals (Sweden)

    Sarah Derks

    2004-01-01

    Full Text Available Methylation‐specific PCR (MSP is a simple, quick and cost‐effective method to analyze the DNA methylation status of virtually any group of CpG sites within a CpG island. The technique comprises two parts: (1 sodium bisulfite conversion of unmethylated cytosine's to uracil under conditions whereby methylated cytosines remains unchanged and (2 detection of the bisulfite induced sequence differences by PCR using specific primer sets for both unmethylated and methylated DNA. This review discusses the critical parameters of MSP and presents an overview of the available MSP variants and the (clinical applications.

  13. Electrocardiografo por computadora

    OpenAIRE

    Tinoco Hernandez, Rosanna; Paredes Bejarano, Margarita; Romero Chaglia, Norman; Yapur Auad, Miguel Eduardo

    2009-01-01

    El presente trabajo trata sobrees el diseño y la implementación de un graficador de señales cardiacas por computadora, para lo cual diseñamos un circuito electrónico capaz de recibir la senal analógica proveniente de la actividad electrica del corazón , amplificarla, y luego convertirla en una señal digital para ser procesada por software y finalmente ser graficada, siendo posible así observar la señal cardiaca en el monitor de un computador como un tren de pulsos. Cabe destacar que par...

  14. pcr

    African Journals Online (AJOL)

    DR. AMINU

    Keywords: Polymerase chain reaction, Diagnosis, Bacteria, Infections. INTRODUCTION ... used to amplify a piece of DNA by in-vitro enzymatic replication (David and .... receiving antimicrobial treatment, or when the causative agents are small ...

  15. Indice por Materias

    Directory of Open Access Journals (Sweden)

    Montoya H Luz Marina

    1982-09-01

    Full Text Available Un índice es una lista de palabras o frases indicadores asociados que permite la ubicación de material al interior de un libro o una publicación, en este caso será por el nombre de la materia.

  16. ANALISIS COSTE-EFECTIVIDAD DE DISTINTOS MÉTODOS DE DIAGNÓSTICO POR IMAGEN DEL TROMBOEMBOLISMO PULMONAR AGUDO

    Directory of Open Access Journals (Sweden)

    Stella Maris Batallés

    2009-01-01

    Full Text Available La técnica diagnóstica óptima para detectar tromboembolismo pulmonar agudo (TEP continúa en discusión. La gammagrafía pulmonar de ventilación/perfusión ha sido el examen preferido durante décadas, pero con el advenimiento de nuevas pruebas de imágenes las posibilidades diagnósticas se ampliaron, siendo necesario evaluarlas desde la perspectiva del coste y de la efectividad. El objetivo de este trabajo fue evaluar distintos métodos de diagnóstico por imagen para detectar TEP agudo para determinar el más coste-efectivo. Métodos. Análisis de coste-efectividad (CE empleando un árbol de decisiones para modelar distintas pruebas (centellograma V/Q, TC helicoidal, angiografía por tomografía computada multidetector (TCMD, resonancia magnética por imágenes (RMI y arteriografía convencional. Se obtuvieron valores de sensibilidad, especificidad, valor predictivo positivo (VPP y negativo (VPN de las pruebas diagnósticas. Resultado medido: "caso detectado de TEP". Los costes evaluados fueron los directos, expresados en euros (t, incluyendo los secundarios a las complicaciones de los métodos diagnósticos. Se realizó un análisis de sensibilidad de una vía para evaluar la robustez de las conclusiones. Resultados. No se eliminaron pruebas por dominancia extendida. La tasa cruda de CE para TCMD fue de 486 t por cada caso de TEP detectado. El coste marginal entre la TC helicoidal y el centellograma V/Q fue de 103 t para detectar 8 casos adicionales de TEP, mientras que el coste marginal entre la TCMD y la TC helicoidal fue de 229 t para detectar un caso adicional de TEP. Conclusiones. La prueba diagnóstica más coste-efectiva fue la TCMD, hallazgo que mostró robustez en el análisis de sensibilidad. Sin embargo, el análisis de C-E incremental nos mostró que la TCMD costó 229 t más respeto a la TC helicoidal para lograr una mínima mejora en la efectividad de la prueba (detección de TEP agudo. El alto valor predictivo negativo de

  17. Apendicitis por Paracoccidioides brasiliensis

    Directory of Open Access Journals (Sweden)

    Ana Beatriz MUÑOZ URRIBARRI

    2006-01-01

    Full Text Available La paracoccidioidomicosis es la micosis más prevalente en Sudamérica. La forma aguda afecta el sistema fagocítico mononuclear de niños y personas inmunocomprometidas. El compromiso gastrointestinal es frecuente y su patogenia implica diseminación hematógena y linfática. La linfadenomegalia abdominal causa obstrucción intestinal y abdomen agudo. En este artículo damos a conocer el caso de un niño con compromiso gastrointestinal por apendicitis. Este es el primer caso reportado de apendicitis por esta patología. (Rev Med Hered 2006;17:58-60.

  18. Avaliação do kit "TF-Test" para o diagnóstico das infecções por parasitas gastrintestinais em ovinos

    Directory of Open Access Journals (Sweden)

    Giuliano Lumina

    2006-08-01

    Full Text Available Este estudo teve como objetivos padronizar o kit TF-Test para a quantificação de ovos de parasitas gastrintestinais de ovinos e compará-lo ao método de Gordon & Whitlock modificado (G&W. Vinte quatro cordeiros confinados foram infectados artificialmente com Haemonchus contortus, durante 12 semanas, até o abate, quando foram colhidas amostras fecais e realizada a identificação e contagem dos parasitas abomasais. Nestes animais, ovos de H. contortus foram detectados em 95,8% das amostras fecais por ambos os testes (P>;0,05. Os coeficientes de correlação (r entre a carga parasitária (CP e os valores de OPG obtidos pelos métodos de G&W e TF-Test foram, respectivamente, de r=0,52 e r=0,51 (dados não transformados e r=0,85 e r=0,87 (dados transformados em log. Outras 100 amostras fecais foram colhidas de ovinos naturalmente infectados. Nas amostras destes animais, os testes G&W e TF-Test propiciaram o diagnóstico de ovos de estrongilídeos em 85% e 86% das amostras, respectivamente (P>;0,05. Pelo TF-Test e pelo G&W, oocistos de Eimeria foram detectados em 33% e em 12% das amostras (P<0,001 e ovos de Strongyloides spp. em 15% e 5% das amostras, respectivamente (P<0,05. Ambos os testes foram precisos para o diagnóstico de estrongilídeos gastrintestinais, porém, o TF-Test foi superior para o diagnóstico de oocistos de Eimeria spp. e de ovos de Strongyloides spp., mas, por outro lado, subestimou o número de ovos de estrongilídeos presente nas amostras.

  19. Two-temperature PCR for Microfluidics

    KAUST Repository

    Kodzius, Rimantas

    2010-05-01

    Since its invention in 1983, polymerase chain reaction (PCR) has been the method of choice for DNA amplification. Successful PCR depends on the optimization of several parameters, which is a cumbersome task due to the many variables (conditions and compon

  20. Two-temperature PCR for Microfluidics

    KAUST Repository

    Kodzius, Rimantas; Chang, Donald Choy; Sheng, Ping; Wen, Weijia; Wu, Jinbo; Xiao, Kang; Yu, Vivian

    2010-01-01

    Since its invention in 1983, polymerase chain reaction (PCR) has been the method of choice for DNA amplification. Successful PCR depends on the optimization of several parameters, which is a cumbersome task due to the many variables (conditions and compon

  1. Apendicitis por Paracoccidioides brasiliensis.

    OpenAIRE

    MUÑOZ URRIBARRI, Ana Beatriz; CHAPARRO DAMMERT, Eduardo; FERRUFINO LLACH, Juan Carlos; VASQUEZ FLORES, Luciola

    2012-01-01

    La paracoccidioidomicosis es la micosis más prevalente en Sudamérica. La forma aguda afecta el sistema fagocítico mononuclear de niños y personas inmunocomprometidas. El compromiso gastrointestinal es frecuente y su patogenia implica diseminación hematógena y linfática. La linfadenomegalia abdominal causa obstrucción intestinal y abdomen agudo. En este artículo damos a conocer el caso de un niño con compromiso gastrointestinal por apendicitis. Este es el primer caso reportado de apendicitis p...

  2. Meningitis por Streptococcus suis

    OpenAIRE

    Geffner Sclarsky, D. E.; Moreno Muñoz, R.; Campillo Alpera, Mª.S.; Pardo Serrano, F.J.; Gómez Gómez, A.; Martínez-Lozano, Mª.D.

    2001-01-01

    La infección humana por Streptococcus suis (S. suis) es una zoonosis, con un riesgo ocupacional conocido y que suele presentarse como meningitis purulenta, que tiene baja mortalidad y frecuentes secuelas de hipoacusia y ataxia. Se han publicado menos de 150 casos humanos desde el informe original de hace 30 años. Hay una reconocida distribución geográfica viviendo la mayoría de los afectados en el norte de Europa y el sudeste Asiático. En España se han comunicado dos pacientes con enfermedad ...

  3. Por mil devaluados pesos

    Directory of Open Access Journals (Sweden)

    Annie Rodríguez Collázos

    2013-03-01

    Full Text Available El estudio de lo popular y lo urbano hasta ahora se ha centrado en el comportamiento y en algunas relaciones de los habitantes con su entorno. “Por mil devaluados pesos. Publicidad popular y urbana”, pretende explorar las formas de publicidad, dispersas en diferentes espacios populares y urbanos en Bogotá, identificando esquemas y formas características de sus propios códigos comunicativos; se centran en un objeto de estudio consistente en las estrategias publicitarias y los códigos comunicativos en los mensajes publicitarios populares en las subculturas de San Victorino, 7 de Agosto y Sanandresito de San José.

  4. Pathogen Causing Disease of Diagnosis PCR Tecnology

    OpenAIRE

    SEVİNDİK, Emre; KIR, A. Çağrı; BAŞKEMER, Kadir; UZUN, Veysel

    2013-01-01

    Polimerase chain reaction (PCR) with which, the development of recombinant DNA tecnology, a technique commonly used in field of moleculer biology and genetic. Duplication of the target DNA is provided with this technique without the need for cloning. Some fungus species, bacteria, viruses constitutent an important group of pathogenicity in human, animals and plants. There are routinely applied types of PCR in the detection of pathogens infections diseases. These Nested- PCR, Real- Time PCR, M...

  5. Principles and technical aspects of PCR amplification

    National Research Council Canada - National Science Library

    Pelt-Verkuil, Elizabeth van; Belkum, Alex van; Hays, John P

    2008-01-01

    ... to illustrate any particularly important concepts or comments. Indeed, all commercial PCR biotechnology companies offer information about their products on internet sites and in online technical manuals. These online resources will be invaluable for any readers requiring more detailed PCR protocols. The authors have provided references for many PCR co...

  6. The PCR revolution: basic technologies and applications

    National Research Council Canada - National Science Library

    Bustin, Stephen A

    2010-01-01

    ... by leading authorities on the many applications of PCR and how this technology has revolutionized their respective areas of interest. This book conveys the ways in which PCR has overcome many obstacles in life science and clinical research and also charts the PCR's development from time-consuming, low throughput, nonquantitative proced...

  7. Análise comparativa entre as metodologias de PCR metilação-específica (MSP, Southern blot (SB e FISH utilizadas no diagnóstico genético molecular de pacientes com suspeita clínica das síndromes de Prader-Willi ou Angelman

    Directory of Open Access Journals (Sweden)

    Bruna Oliveira

    2016-08-01

    Full Text Available Introdução: Prader-Willi (SPW e Angelman (SA são síndromes clinicamente distintas, causadas pela perda de expressão de genes na região cromossômica 15q11.2-q13, de origem paterna ou materna, respectivamente. Ambas compartilham os mesmos métodos diagnósticos. Nossos objetivos foram: a analisar por PCR metilação-específica (MSP pacientes com suspeita clínica de SPW/SA; b comparar resultados de diferentes metodologias de diagnóstico molecular; c aplicar a técnica MSP na rotina assistencial de pacientes encaminhados ao Serviço de Genética Médica/Hospital de Clínicas de Porto Alegre (SGM/HCPA. Métodos: Foram analisados 123 pacientes com suspeita clínica de SPW (n = 71 ou SA (n = 52 por MSP. Desses, 79 possuíam análise prévia por hibridação in situ fluorescente (FISH e/ou Southern blot (SB. Resultados: Foram detectados 21 casos positivos – 15 de SPW (12,19% e 6 de SA (4,88%. Nove pacientes tiveram etiologia molecular determinada, sendo sete com diagnóstico de SPW (quatro dissomias uniparentais – UPD15 materna – e três deleções na região 15q11-13 e dois com diagnóstico de SA (um com UPD15 paterna e um com deleção na região 15q11-13. Foram observados resultados equivalentes entre MSP e SB e resultados discrepantes entre MSP e FISH (n = 4. Foram padronizados dois protocolos de MSP para confirmação dos resultados e controle interno de qualidade. Conclusão: O perfil de detecção de cada técnica varia de acordo com o mecanismo etiológico presente. A análise por MSP detecta alterações no padrão de metilação geradas por deleção, UPD e defeitos de imprinting, sem identificar o mecanismo etiológico responsável. Contudo, mostrou ser eficiente para confirmação do diagnóstico clínico e screening dos pacientes com suspeita clínica sugestiva de SPW e SA. Diante de resultados positivos, é importante a identificação do mecanismo molecular subjacente para correlação genótipo-fenótipo e determina

  8. Sepsis por shigella flexneri

    Directory of Open Access Journals (Sweden)

    César Cabrera C

    2005-04-01

    Full Text Available Se presenta un caso raro de sepsis por Shigella flexneri en una paciente de 45 años de edad quien estando hospitalizada para el estudio de un tumor cerebral, requirió el uso de manitol y dosis altas de corticoides; luego de ello presenta deposiciones líquidas con moco y sangre, desarrolla síndrome de respuesta inflamatoria sistémica, luego se aísla Shigella flexneri en el hemocultivo; recibió tratamiento antibiótico con ciprofloxacina. Se describen las características del caso y se comenta de acuerdo con la revisión de literatura.

  9. Real-Time PCR (qPCR) Primer Design Using Free Online Software

    Science.gov (United States)

    Thornton, Brenda; Basu, Chhandak

    2011-01-01

    Real-time PCR (quantitative PCR or qPCR) has become the preferred method for validating results obtained from assays which measure gene expression profiles. The process uses reverse transcription polymerase chain reaction (RT-PCR), coupled with fluorescent chemistry, to measure variations in transcriptome levels between samples. The four most…

  10. Detection of adenoviruses in shellfish by means of conventional-PCR, nested-PCR, and integrated cell culture PCR (ICC/PCR).

    Science.gov (United States)

    Rigotto, C; Sincero, T C M; Simões, C M O; Barardi, C R M

    2005-01-01

    We tested three PCR based methodologies to detect adenoviruses associated with cultivated oysters. Conventional-PCR, nested-PCR, and integrated cell culture-PCR (ICC/PCR) were first optimized using oysters seeded with know amounts of Adenovirus serotype 5 (Ad5). The maximum sensitivity for Ad5 detection was determined for each method, and then used to detect natural adenovirus contamination in oysters from three aquiculture farms in Florianopolis, Santa Catarina State, Brazil, over a period of 6 months. The results showed that the nested-PCR was more sensitive (limit of detection: 1.2 PFU/g of tissue) than conventional-PCR and ICC-PCR (limit of detection for both: 1.2 x 10(2)PFU/g of tissue) for detection of Ad5 in oyster extracts. Nested-PCR was able to detect 90% of Ad5 contamination in harvested oyster samples, while conventional-PCR was unable to detect Ad5 in any of the samples. The present work suggests that detection of human adenoviruses can be used as a tool to monitor the presence of human viruses in marine environments where shellfish grow, and that nested-PCR is the method of choice.

  11. Portable exhausters POR-004 SKID B, POR-005 SKID C, POR-006 SKID D storage plan

    International Nuclear Information System (INIS)

    Nelson, O.D.; Keller, G.M.

    1997-01-01

    This document provides a storage plan for portable exhausters POR-004 SKID B, POR-005 SKID C, AND POR-006 SKID D. The exhausters will be stored until they are needed by the TWRS (Tank Waste Remediation Systems) Saltwell Pumping Program. The storage plan provides criteria for portable exhauster storage, periodic inspections during storage, and retrieval from storage

  12. SEPARATION OF X-BEARING BOVINE SPERM BY CENTRIFUGATION IN CONTINUOUS PERCOLL AND OPTIPREP DENSITY GRADIENT: EFFECT IN SPERM VIABILITY AND IN VITRO EMBRYO PRODUCTION SEPARAÇÃO DE ESPERMATOZOIDES PORTADORES DO CROMOSSOMO X BOVINO POR CENTRIFUGAÇÃO EM GRADIENTE DE DENSIDADE CONTÍNUO DE PERCOLL E OPTIPREP: EFEITO SOBRE A VIABILIDADE ESPERMÁTICA E NA PRODUÇÃO IN VITRO DE EMBRIÕES

    Directory of Open Access Journals (Sweden)

    Aline Costa Lucio

    2009-07-01

    Full Text Available

    The aim of this study was to separate X-bearing bovine sperm by continuous Percoll and OptiPrep density gradients and to validate the sexing of resultant in vitro produced embryos by Polimerase Chain Reaction (PCR. Frozen/thawed sperm was layered on density gradients which were previously prepared in polystyrene tubes, 24 h before procedures and maintained at 4 °C. The tubes were centrifuged at 500 x g for 15 min at 22 °C. Supernatants were gently aspirated and the sperm recovered from the bottom of the tubes. Viability and integrity of sperm were evaluated by Trypan Blue/Giemsa stain. Cleavage and blastocyst rates were determined by in vitro production of embryos and PCR was performed for identification of the embryos’ genetic sex. No damage in viability and acrossomal integrity and in cleavage and blastocyst rates was found in the Percoll and OptiPrep treatment compared to the non-centrifuged group (P>0.05. The percentage of female embryos in the Percoll and OptiPrep group was 63.0 and 47.6%, respectively. The female embryos in control group were 48.7%. A sexual deviation in the Percoll density gradient was achieved without reduction of sperm viability and in vitro production rates.

    KEY WORDS: Bovine, centrifugation, in vitro production of embryos, PCR, X-bearing sperm.

    O objetivo deste estudo foi separar espermatozoides bovinos portadores do cromossomo X pela centrifugação em gradiente de densidade contínuo de Percoll e OptiPrep, e validar a sexagem pela reação em cadeia da polimerase (PCR, dos embriões produzidos in vitro. Para a sexagem, espermatozoides descongelados foram depositados nos gradientes de densidade, previamente preparados, em tubos de poliestireno, 24 horas antes da sexagem e mantidos a 4°C. Centrifugou-se a 500 x g por quinze minutos a 22°C. Os sobrenadantes foram aspirados, e os espermatozoides recuperados do

  13. Control Multivariable por Desacoplo

    Directory of Open Access Journals (Sweden)

    Fernando Morilla

    2013-01-01

    Full Text Available Resumen: La interacción entre variables es una característica inherente de los procesos multivariables, que dificulta su operación y el diseño de sus sistemas de control. Bajo el paradigma de Control por desacoplo se agrupan un conjunto de metodologías, que tradicionalmente han estado orientadas a eliminar o reducir la interacción, y que recientemente algunos investigadores han reorientado con objetivos de solucionar un problema tan complejo como es el control multivariable. Parte del material descrito en este artículo es bien conocido en el campo del control de procesos, pero la mayor parte de él son resultados de varios años de investigación de los autores en los que han primado la generalización del problema, la búsqueda de soluciones de fácil implementación y la combinación de bloques elementales de control PID. Esta conjunción de intereses provoca que no siempre se pueda conseguir un desacoplo perfecto, pero que sí se pueda conseguir una considerable reducción de la interacción en el nivel básico de la pirámide de control, en beneficio de otros sistemas de control que ocupan niveles jerárquicos superiores. El artículo resume todos los aspectos básicos del Control por desacoplo y su aplicación a dos procesos representativos: una planta experimental de cuatro tanques acoplados y un modelo 4×4 de un sistema experimental de calefacción, ventilación y aire acondicionado. Abstract: The interaction between variables is inherent in multivariable processes and this fact may complicate their operation and control system design. Under the paradigm of decoupling control, several methodologies that traditionally have been addressed to cancel or reduce the interactions are gathered. Recently, this approach has been reoriented by several researchers with the aim to solve such a complex problem as the multivariable control. Parts of the material in this work are well known in the process control field; however, most of them are

  14. SIMPLIFIED DIAGNOSIS OF MALARIA INFECTION: GFM/PCR/ELISA A SIMPLIFIED NUCLEIC ACID AMPLIFICATION TECHNIQUE BY PCR/ELISA

    Directory of Open Access Journals (Sweden)

    Ricardo Luiz Dantas MACHADO

    1998-09-01

    Full Text Available We report an adaptation of a technique for the blood sample collection (GFM as well as for the extraction and amplification of Plasmodium DNA for the diagnosis of malaria infection by the PCR/ELISA. The method of blood sample collection requires less expertise and saves both time and money, thus reducing the cost by more than half. The material is also suitable for genetic analysis in either fresh or stored specimens prepared by this method.Relatamos a adaptação de uma técnica para coleta de amostras (MFV e outra para extração, amplificação de DNA de parasitas da malária para diagnóstico por PCR/ELISA. O método de coleta de amostras requer menos habilidade e economisa tempo e dinheiro, assim reduzindo a mais da metade o custo. O material é também adequado para análise genética em especimens frescos ou estocados, preparados por este método.

  15. Predominant porB1A and porB1B genotypes and correlation of gene mutations with drug resistance in Neisseria gonorrhoeae isolates in Eastern China

    Directory of Open Access Journals (Sweden)

    Tang Renxian

    2010-11-01

    Full Text Available Abstract Background Variations of porB1A and porB1B genes and their serotypes exist in Neisseria gonorrhoeae isolates from different geographical areas, and some site mutations in the porB1B gene correlate with drug resistance. Methods The β-lactamase production of N. gonorrhoeae isolates was determined by paper acidometric test and nitrocefin discs. The porB1A and porB1B genes of 315 non-penicillinase-producting N. gonorrhoeae (non-PPNG strains were amplified by PCR for sequencing to determine serotypes and site mutations. A duplex PCR was designed to simultaneously detect both porB1A and porB1B genes. Penicillin and tetracycline resistance was assessed by an in vitro drug sensitivity test. Results Of the N. gonorrhoeae isolates, 31.1% tested positive for porB1A and 68.9% for porB1B genes. All the 98 porB1A+ isolates belonging to IA6 serotype with either no mutation at the 120 and 121 sites (88.8% or a D120G (11.2% mutation and were no resistance to both penicillin and tetracycline. Among the 217 porB1B+ isolates, 26.7%, 22.6% and 11.5% belonged to IB3, IB3/6 and IB4 serotypes, respectively. Particularly, two novel chimeric serotypes, IB3/6-IB2 and IB2-IB4-IB2, were found in 77 and 8 porB1B+ isolates. Two hundred and twelve (97.7% of the porB1B+ isolates were presented G120 and/or A121 mutations with 163 (76.9% at both sites. Interestingly, within the 77 porB1B+ isolates belonging to IB3/6-IB2 serotype, 15 were discovered to possess novel deletions at both A121 and N122 sites. All the replacement mutations at these sites in PorB1B were correlated with resistance and the deletion mutation showed the highest resistance. Conclusion N. gonorrhoeae isolates circulating in Eastern China include a sole PorB1A serotype (IA6 and five PorB1B serotypes. Multiple mutations in porB1B genes, including novel A121 and N122 deletions, are correlated with high levels of penicillin and tetracycline resistance.

  16. Application of reverse transcription-PCR and real-time PCR in nanotoxicity research.

    Science.gov (United States)

    Mo, Yiqun; Wan, Rong; Zhang, Qunwei

    2012-01-01

    Reverse transcription-polymerase chain reaction (RT-PCR) is a relatively simple and inexpensive technique to determine the expression level of target genes and is widely used in biomedical science research including nanotoxicology studies for semiquantitative analysis. Real-time PCR allows for the detection of PCR amplification in the exponential growth phase of the reaction and is much more quantitative than traditional RT-PCR. Although a number of kits and reagents for RT-PCR and real-time PCR are commercially available, the basic principles are the same. Here, we describe the procedures for total RNA isolation by using TRI Reagent, for reverse transcription (RT) by M-MLV reverse transcriptase, and for PCR by GoTaq(®) DNA Polymerase. And real-time PCR will be performed on an iQ5 multicolor real-time PCR detection system by using iQ™ SYBR Green Supermix.

  17. Abortos por Neosporacaninum em bovinos do sul de Minas Gerais

    Directory of Open Access Journals (Sweden)

    Débora R. Orlando

    2013-11-01

    Full Text Available Este estudo avaliou a participação de Neospora caninum em casos de abortos em bovinos provenientes de propriedades rurais da região sul de Minas Gerais por meio de análises histopatológicas, imuno-histoquímicas (IHQ e pela reação em Cadeia de Polimerase (PCR. O material utilizado foi obtido de um estudo retrospectivo de casos de aborto recebidos pelo Setor de Patologia Veterinária da Universidade Federal de Lavras e de fetos necropsiados durante os anos de 2011 a 2013. De 60 fetos estudados, 30 (50% tinham lesões microscópicas. Destes, 19 (63% apresentaram lesões compatíveis com aborto por N. caninum, caracterizadas principalmente por encefalite não supurativa multifocal, necrose e gliose multifocal, assim como, miocardite e miosite não supurativa. Em 14 fetos chegou-se ao diagnóstico definitivo. Destes, cinco tiveram sua confirmação somente pela marcação IHQ e cinco foram positivos somente na PCR. Quatro fetos foram positivos tanto na IHQ quanto na PCR. Cinco fetos, provenientes do estudo retrospectivo apresentaram lesões compatíveis com N. caninum, mas a presença do protozoário não foi confirmada pela marcação IHQ. Os achados demonstram que o N. caninum é um importante agente associado ao aborto em bovinos na região sul de Minas Gerais. Para tanto, além das lesões microscópicas a associação entre a IHQ e a técnica de PCR foi essencial para a confirmação do diagnóstico.

  18. Pitfalls in PCR troubleshooting: Expect the unexpected?

    Directory of Open Access Journals (Sweden)

    Livia Schrick

    2016-01-01

    Full Text Available PCR is a well-understood and established laboratory technique often used in molecular diagnostics. Huge experience has been accumulated over the last years regarding the design of PCR assays and their set-up, including in-depth troubleshooting to obtain the optimal PCR assay for each purpose. Here we report a PCR troubleshooting that came up with a surprising result never observed before. With this report we hope to sensitize the reader to this peculiar problem and to save troubleshooting efforts in similar situations, especially in time-critical and ambitious diagnostic settings.

  19. Absolute quantification by droplet digital PCR versus analog real-time PCR

    Science.gov (United States)

    Hindson, Christopher M; Chevillet, John R; Briggs, Hilary A; Gallichotte, Emily N; Ruf, Ingrid K; Hindson, Benjamin J; Vessella, Robert L; Tewari, Muneesh

    2014-01-01

    Nanoliter-sized droplet technology paired with digital PCR (ddPCR) holds promise for highly precise, absolute nucleic acid quantification. Our comparison of microRNA quantification by ddPCR and real-time PCR revealed greater precision (coefficients of variation decreased by 37–86%) and improved day-to-day reproducibility (by a factor of seven) of ddPCR but with comparable sensitivity. When we applied ddPCR to serum microRNA biomarker analysis, this translated to superior diagnostic performance for identifying individuals with cancer. PMID:23995387

  20. Advantages and limitations of quantitative PCR (Q-PCR)-based approaches in microbial ecology.

    Science.gov (United States)

    Smith, Cindy J; Osborn, A Mark

    2009-01-01

    Quantitative PCR (Q-PCR or real-time PCR) approaches are now widely applied in microbial ecology to quantify the abundance and expression of taxonomic and functional gene markers within the environment. Q-PCR-based analyses combine 'traditional' end-point detection PCR with fluorescent detection technologies to record the accumulation of amplicons in 'real time' during each cycle of the PCR amplification. By detection of amplicons during the early exponential phase of the PCR, this enables the quantification of gene (or transcript) numbers when these are proportional to the starting template concentration. When Q-PCR is coupled with a preceding reverse transcription reaction, it can be used to quantify gene expression (RT-Q-PCR). This review firstly addresses the theoretical and practical implementation of Q-PCR and RT-Q-PCR protocols in microbial ecology, highlighting key experimental considerations. Secondly, we review the applications of (RT)-Q-PCR analyses in environmental microbiology and evaluate the contribution and advances gained from such approaches. Finally, we conclude by offering future perspectives on the application of (RT)-Q-PCR in furthering understanding in microbial ecology, in particular, when coupled with other molecular approaches and more traditional investigations of environmental systems.

  1. Bioinformatic tools for PCR Primer design

    African Journals Online (AJOL)

    ES

    reaction (PCR), oligo hybridization and DNA sequencing. Proper primer design is actually one of the most important factors/steps in successful DNA sequencing. Various bioinformatics programs are available for selection of primer pairs from a template sequence. The plethora programs for PCR primer design reflects the.

  2. [E-MTAB-587] PCR_artifacts

    NARCIS (Netherlands)

    Muino Acuna, J.M.

    2011-01-01

    WARNING: This library was yield low amount of material and it was over-amplified by PCR. This libraries are used study the robustness of several statitical methods against PCR artifacts. ChIP experiments were performed on Arabidopsis wildtype inflorescences using an antibody raised against a

  3. Digital PCR for detection of citrus pathogens

    Science.gov (United States)

    Citrus trees are often infected with multiple pathogens of economic importance, especially those with insect or mite vectors. Real-time/quantitative PCR (qPCR) has been used for high-throughput detection and relative quantification of pathogens; however, target reference or standards are required. I...

  4. Testing for Genetically Modified Foods Using PCR

    Science.gov (United States)

    Taylor, Ann; Sajan, Samin

    2005-01-01

    The polymerase chain reaction (PCR) is a Nobel Prize-winning technique that amplifies a specific segment of DNA and is commonly used to test for the presence of genetic modifications. Students use PCR to test corn meal and corn-muffin mixes for the presence of a promoter commonly used in genetically modified foods, the cauliflower mosaic virus 35S…

  5. Validation of RNAi by real time PCR

    DEFF Research Database (Denmark)

    Josefsen, Knud; Lee, Ying Chiu

    2011-01-01

    Real time PCR is the analytic tool of choice for quantification of gene expression, while RNAi is concerned with downregulation of gene expression. Together, they constitute a powerful approach in any loss of function studies of selective genes. We illustrate here the use of real time PCR to verify...

  6. PCR specific for Actinobacillus pleuropneumoniae serotype 3

    DEFF Research Database (Denmark)

    Zhou, L.; Jones, S.C.P.; Angen, Øystein

    2008-01-01

    , but the method has liminations, for example, cross-reactions between serotypes 3, 6, and 8. This study describes the development of a serotype 3-specific PCR, based on the capsule locus, which can be used in a multiplex format with the organism's specific gene apxIV. The PCR test was evaluated on 266 strains...

  7. Polymerase chain reaction methods (PCR in agrobiotechnology

    Directory of Open Access Journals (Sweden)

    Taški-Ajduković Ksenija

    2006-01-01

    Full Text Available The agricultural biotechnology applies polymerase chain reaction (PCR technology at numerous steps throughout product development. The major uses of PCR technology during product development include gene discovery and cloning, vector construction, transformant identification, screening and characterization as well as seed quality control. Commodity and food companies as well as testing laboratories rely on PCR technology to verify the presence or absence of genetically modification (GM in a product or to quantify the amount of GM material present in the product. This article describes the fundamental elements of PCR analysis and its application to the testing of grains and highlights some of areas to which attention must be paid in order to produce reliable test results. The article also discuses issues related to the analysis of different matrixes and the effect they may have on the accuracy of the PCR analytical results.

  8. Mathematical analysis of the real time array PCR (RTA PCR) process

    NARCIS (Netherlands)

    Dijksman, Johan Frederik; Pierik, A.

    2012-01-01

    Real time array PCR (RTA PCR) is a recently developed biochemical technique that measures amplification curves (like with quantitative real time Polymerase Chain Reaction (qRT PCR)) of a multitude of different templates in a sample. It combines two different methods in order to profit from the

  9. Real-time PCR in virology.

    Science.gov (United States)

    Mackay, Ian M; Arden, Katherine E; Nitsche, Andreas

    2002-03-15

    The use of the polymerase chain reaction (PCR) in molecular diagnostics has increased to the point where it is now accepted as the gold standard for detecting nucleic acids from a number of origins and it has become an essential tool in the research laboratory. Real-time PCR has engendered wider acceptance of the PCR due to its improved rapidity, sensitivity, reproducibility and the reduced risk of carry-over contamination. There are currently five main chemistries used for the detection of PCR product during real-time PCR. These are the DNA binding fluorophores, the 5' endonuclease, adjacent linear and hairpin oligoprobes and the self-fluorescing amplicons, which are described in detail. We also discuss factors that have restricted the development of multiplex real-time PCR as well as the role of real-time PCR in quantitating nucleic acids. Both amplification hardware and the fluorogenic detection chemistries have evolved rapidly as the understanding of real-time PCR has developed and this review aims to update the scientist on the current state of the art. We describe the background, advantages and limitations of real-time PCR and we review the literature as it applies to virus detection in the routine and research laboratory in order to focus on one of the many areas in which the application of real-time PCR has provided significant methodological benefits and improved patient outcomes. However, the technology discussed has been applied to other areas of microbiology as well as studies of gene expression and genetic disease.

  10. Infecciones por bacterias poco comunes y oncogénesis bacteriana

    Directory of Open Access Journals (Sweden)

    Horacio A. Lopardo

    Full Text Available La recuperación de algunos microorganismos de aislamiento esporádico en el laboratorio de microbiología clínica podría significar la existencia de algún defecto inmunitarioespecial en el paciente. Por ejemplo, se ha descrito una importante correlación entre Clostridium septicum y carcinoma de colon, y se han visto relacionadas con leucemias y linfomas a especies que aparecen casi siempre como contaminantes (Bacillus spp., Corynebacterium spp. y a otras raramente aisladas en otros contextos (Capnocytophaga spp.. Hay bacterias que se aíslan casi exclusivamente de pacientes con sida (Rhodococcus equi. Se ha observado una mayor frecuencia de infecciones por Campylobacter spp., Aeromonas spp. y estreptococos del grupo G y del grupo mitis en individuos con algún tipo de cáncer que en el resto de los pacientes. También hay bacterias que son marcadoras de algún cáncer no detectado o que afectan más a pacientes neutropénicos que a individuos normoinmunes. La alteración de la reacción inflamatoria, la linfoproliferación mediada por antígenos bacterianos y la inducción de hormonas que aumentan la proliferación de las células epiteliales podrían ser causas de la oncogénesis bacteriana. Los ejemplos clásicos son el adenocarcinoma gástrico inducido por Helicobacter pylori, la asociación de la bacteriemia por estreptococos del grupo bovis y el cáncer de colon y los linfomas de tejido linfoide asociado a mucosas (MALT en vinculación con especies de Helicobacter (MALT gástricos y con Chlamydophila spp. (MALT oculares. El aislamiento de alguno de estos patógenos debería ser un llamado de atención para inducir al estudio de alguna enfermedad maligna.

  11. Assessment of the real-time PCR and different digital PCR platforms for DNA quantification.

    Science.gov (United States)

    Pavšič, Jernej; Žel, Jana; Milavec, Mojca

    2016-01-01

    Digital PCR (dPCR) is beginning to supersede real-time PCR (qPCR) for quantification of nucleic acids in many different applications. Several analytical properties of the two most commonly used dPCR platforms, namely the QX100 system (Bio-Rad) and the 12.765 array of the Biomark system (Fluidigm), have already been evaluated and compared with those of qPCR. However, to the best of our knowledge, direct comparison between the three of these platforms using the same DNA material has not been done, and the 37 K array on the Biomark system has also not been evaluated in terms of linearity, analytical sensitivity and limit of quantification. Here, a first assessment of qPCR, the QX100 system and both arrays of the Biomark system was performed with plasmid and genomic DNA from human cytomegalovirus. With use of PCR components that alter the efficiency of qPCR, each dPCR platform demonstrated consistent copy-number estimations, which indicates the high resilience of dPCR. Two approaches, one considering the total reaction volume and the other considering the effective reaction size, were used to assess linearity, analytical sensitivity and variability. When the total reaction volume was considered, the best performance was observed with qPCR, followed by the QX100 system and the Biomark system. In contrast, when the effective reaction size was considered, all three platforms showed almost equal limits of detection and variability. Although dPCR might not always be more appropriate than qPCR for quantification of low copy numbers, dPCR is a suitable method for robust and reproducible quantification of viral DNA, and a promising technology for the higher-order reference measurement method.

  12. Diagnóstico temprano en un brote epidémico del virus Dengue en Piura usando RT-PCR y nested-PCR

    Directory of Open Access Journals (Sweden)

    Oscar Nolasco

    1997-07-01

    Full Text Available Un test de diagnóstico temprano (RT-PCR y Nested-PCR fue evaluado y comparado con métodos convencionales (cultivo in vitro, IFI y MAC-ELISA. Treinta y cuatro sueros de pacientes correspondientes de un brote epidémico de la costa norte peruana (Mancora, Piura en mayo de 1997 fueron incluidos en este estudio. Todos los sueros fueron obtenidos de pacientes que presentaron en los primeros cinco días manifestaciones clínicas siendo diagnosticados luego como dengue serotipo 1. Asimismo, RT-PCR permitió diagnosticar 82% de los sueros (28/34, sin embargo Mac-ELISA y cultivo in vitro reconocieron unicamente 41% de los sueros (14/34 y 38% de los sueros (13/34 respectivamente. Por lo tanto, el uso de esta herramienta molecular (RT-PCR y Nested-PCR permitiró dar un diagnóstico temprano a estos pacientes y actuar inmediatamente ante la presencia de un brote epidémico.

  13. Material Biocompatibility for PCR Microfluidic Chips

    KAUST Repository

    Kodzius, Rimantas; Chang, Donald Choy; Gong, Xiuqing; Wen, Weijia; Wu, Jinbo; Xiao, Kang; Yi, Xin

    2010-01-01

    As part of the current miniaturization trend, biological reactions and processes are being adapted to microfluidics devices. PCR is the primary method employed in DNA amplification, its miniaturization is central to efforts to develop portable devices for diagnostics and testing purposes. A problem is the PCR-inhibitory effect due to interaction between PCR reagents and the surrounding environment, which effect is increased in high-surface-are-to-volume ration microfluidics. In this study, we evaluated the biocompatibility of various common materials employed in the fabrication of microfluidic chips, including silicon, several kinds of silicon oxide, glasses, plastics, wax, and adhesives. Two-temperature PCR was performed with these materials to determine their PCR-inhibitory effect. In most of the cases, addition of bovine serum albumin effectively improved the reaction yield. We also studied the individual PCR components from the standpoint of adsorption. Most of the materials did not inhibit the DNA, whereas they did show noticeable interaction with the DNA polymerase. Our test, instead of using microfluidic devices, can be easily conducted in common PCR tubes using a standard bench thermocycler. Our data supports an overview of the means by which the materials most bio-friendly to microfluidics can be selected.

  14. Material Biocompatibility for PCR Microfluidic Chips

    KAUST Repository

    Kodzius, Rimantas

    2010-04-23

    As part of the current miniaturization trend, biological reactions and processes are being adapted to microfluidics devices. PCR is the primary method employed in DNA amplification, its miniaturization is central to efforts to develop portable devices for diagnostics and testing purposes. A problem is the PCR-inhibitory effect due to interaction between PCR reagents and the surrounding environment, which effect is increased in high-surface-are-to-volume ration microfluidics. In this study, we evaluated the biocompatibility of various common materials employed in the fabrication of microfluidic chips, including silicon, several kinds of silicon oxide, glasses, plastics, wax, and adhesives. Two-temperature PCR was performed with these materials to determine their PCR-inhibitory effect. In most of the cases, addition of bovine serum albumin effectively improved the reaction yield. We also studied the individual PCR components from the standpoint of adsorption. Most of the materials did not inhibit the DNA, whereas they did show noticeable interaction with the DNA polymerase. Our test, instead of using microfluidic devices, can be easily conducted in common PCR tubes using a standard bench thermocycler. Our data supports an overview of the means by which the materials most bio-friendly to microfluidics can be selected.

  15. Comparison between digital PCR and real-time PCR in detection of Salmonella typhimurium in milk.

    Science.gov (United States)

    Wang, Meng; Yang, Junjie; Gai, Zhongtao; Huo, Shengnan; Zhu, Jianhua; Li, Jun; Wang, Ranran; Xing, Sheng; Shi, Guosheng; Shi, Feng; Zhang, Lei

    2018-02-02

    As a kind of zero-tolerance foodborne pathogens, Salmonella typhimurium poses a great threat to quality of food products and public health. Hence, rapid and efficient approaches to identify Salmonella typhimurium are urgently needed. Combined with PCR and fluorescence technique, real-time PCR (qPCR) and digital PCR (ddPCR) are regarded as suitable tools for detecting foodborne pathogens. To compare the effect between qPCR and ddPCR in detecting Salmonella typhimurium, a series of nucleic acid, pure strain culture and spiking milk samples were applied and the resistance to inhibitors referred in this article as well. Compared with qPCR, ddPCR exhibited more sensitive (10 -4 ng/μl or 10 2 cfu/ml) and less pre-culturing time (saving 2h). Moreover, ddPCR had stronger resistance to inhibitors than qPCR, yet absolute quantification hardly performed when target's concentration over 1ng/μl or 10 6 cfu/ml. This study provides an alternative strategy in detecting foodborne Salmonella typhimurium. Copyright © 2018 Elsevier B.V. All rights reserved.

  16. Avaliação da morfologia interna de sementes de Acca sellowiana O. Berg por meio de análise de imagens

    Directory of Open Access Journals (Sweden)

    Vanessa Neumann Silva

    2013-12-01

    Full Text Available Acca sellowiana O. (Berg Burret é uma fruteira nativa da região Sul do Brasil e do Uruguai, que apresenta grande potencial de uso na recuperação de áreas degradadas. O objetivo deste trabalho foi avaliar a morfologia interna de sementes de Acca sellowiana O. Berg por meio da análise de imagens de raios X e relacionar os resultados com a germinação das sementes. Sementes de Acca sellowiana O. Berg, representadas por três lotes, foram analisadas pelo teste de raios X e, posteriormente, conduzidas ao teste de germinação, com avaliação aos 44 dias após a semeadura. As imagens de raios X foram analisadas com o software ImageJ. A análise das imagens radiográficas de sementes de Acca sellowiana permite a mensuração das áreas internas livres, assim como a determinação da relação entre estas e a germinação. Danos internos detectados por meio de raios X afetam a germinação das sementes.

  17. Análise da contaminação de chupetas por enteroparasitas e fungos em escola de ensino fundamental

    Directory of Open Access Journals (Sweden)

    Paola de Oliveira Abreu

    2016-12-01

    Full Text Available Introdução: Como qualquer outro objeto levado à boca, a chupeta apresenta-se como um reservatório potencial de microorganismos. Através dessa pesquisa, busca-se avaliar as chupetas utilizadas por crianças, com o propósito de detectar os microorganismos mais prevalentes e, a partir disso, planejar estratégias de prevenção, controle e redução de possíveis enfermidades na infância. Métodos: Foi realizado um estudo prospectivo, descritivo e observacional com fontes de dados primários, obtido por meio da análise de chupetas. O grupo em estudo era constituído por crianças de ambos os sexos, com idade entre um a seis anos, matriculados em escola municipal de ensino infantil, em Santa Cruz do Sul. Resultados: Foram analisadas 72 chupetas de crianças com idade média de 3,46 anos, sendo 51,39%, do sexo masculino. Em quatro amostras foram encontradas Candida albicans, não sendo detectados enteroparasitas. Foi observado que 9,72% das chupetas nunca são limpas antes de serem oferecidas à criança e mais de 20% delas não são guardadas em local adequado. A água consumida pela criança na própria residência foi oriunda da torneira em 70,83% e filtrada em 13,88% dos casos. Na escola, a água consumida era apenas da torneira. Observou-se que 9,32% das mães tinham conhecimento vago sobre a transmissão de parasitas pela chupeta. Conclusões: A prevalência de enteropatógenos detectados nas chupetas estudadas foi menor do que a descrita na literatura, porém o estudo demonstra a necessidade de um melhor esclarecimento aos pais quanto aos cuidados para prevenção de enfermidades e sobre os malefícios que o uso da chupeta pode ocasionar.

  18. Real-time PCR in virology

    OpenAIRE

    Mackay, Ian M.; Arden, Katherine E.; Nitsche, Andreas

    2002-01-01

    The use of the polymerase chain reaction (PCR) in molecular diagnostics has increased to the point where it is now accepted as the gold standard for detecting nucleic acids from a number of origins and it has become an essential tool in the research laboratory. Real-time PCR has engendered wider acceptance of the PCR due to its improved rapidity, sensitivity, reproducibility and the reduced risk of carry-over contamination. There are currently five main chemistries used for the detection of P...

  19. Porøse materialer

    DEFF Research Database (Denmark)

    Hansen, Ernst Jan de Place

    2000-01-01

    Dette undervisningsnotat er en samling af noter, der refererer til den indledende del af kurset Materialmekanik og Porøse materailer på Insitut for Bærende Konstruktiner og Materialer (BKM).......Dette undervisningsnotat er en samling af noter, der refererer til den indledende del af kurset Materialmekanik og Porøse materailer på Insitut for Bærende Konstruktiner og Materialer (BKM)....

  20. Detección y estudio mediante Fluorescencia Inducida por Láser de radicales libres formados por Disociación Multifotónica Infrarroja

    Science.gov (United States)

    Santos, M.; Díaz, L.; Torresano, J. A.; Rubio, L.; Samoudi, B.

    Una de las principales aplicaciones actuales de los procesos de disociación multifotónica inducidos por radiación láser infrarroja (DMI) es la producción de radiales libres, con el fin de estudiar sus propiedades cinéticas y espectroscópicas. La disociación de moléculas poliatómicas en el IR con láseres de CO2 tiene lugar desde la superficie de energía molecular mas baja y conduce generalmente a la formación de fragmentos en el estado electrónico fundamental, con diversos grados de excitación vibracional. En el Grupo de Procesos Multifotónicos del Instituto de Estructura de la Materia del C.S.I.C. hemos puesto a punto la técnica de Fluorescencia Inducida por Láser (LIF) para la detección y análisis en tiempo real de los fragmentos producidos en la DMI inducida mediante uno o dos campos láseres de diferentes longitudes de onda. Objetivos de nuestro trabajo han sido el estudio de los canales de disociación mayoritarios y de las especies transitoria producidas, así como de la distribución de energía interna con que éstas son generadas. En particular hemos detectado mediante LIF las especies: C2, CF, CH, SiH2, CF2, CH2, SiHCl, y CF3 a partir de la disociación de, entre otras, las siguientes moléculas: C2H3Br, C3F6, C4H8Si, C2H5ClSi y CH5ClSi. En este trabajo presentamos algunos de los resultados obtenidos mediante el estudio por LIF de estos radicales: estudio temporal de la señal LIF obtenida con determinación de tiempos de vida, espectros de excitación y fluorescencia, temperaturas vibracionales de formación, variación de la intensidad LIF con el tiempo de retraso entre los láseres de disociación y prueba, etc.

  1. Calibrated user-friendly reverse transcriptase-PCR assay

    DEFF Research Database (Denmark)

    Bor, M V; Sørensen, B S; Rammer, P

    1998-01-01

    We report a competitive reverse transcriptase-PCR (RT-PCR) assay and a calibrated user-friendly RT-PCR assay (CURT-PCR) for epidermal growth factor receptor (EGFR) mRNA. A calibrator was prepared from isolated rat liver RNA, and the amount of EGFR mRNA was determined by competitive RT-PCR. In CUR...

  2. Transgene detection by digital droplet PCR.

    Directory of Open Access Journals (Sweden)

    Dirk A Moser

    Full Text Available Somatic gene therapy is a promising tool for the treatment of severe diseases. Because of its abuse potential for performance enhancement in sports, the World Anti-Doping Agency (WADA included the term 'gene doping' in the official list of banned substances and methods in 2004. Several nested PCR or qPCR-based strategies have been proposed that aim at detecting long-term presence of transgene in blood, but these strategies are hampered by technical limitations. We developed a digital droplet PCR (ddPCR protocol for Insulin-Like Growth Factor 1 (IGF1 detection and demonstrated its applicability monitoring 6 mice injected into skeletal muscle with AAV9-IGF1 elements and 2 controls over a 33-day period. A duplex ddPCR protocol for simultaneous detection of Insulin-Like Growth Factor 1 (IGF1 and Erythropoietin (EPO transgenic elements was created. A new DNA extraction procedure with target-orientated usage of restriction enzymes including on-column DNA-digestion was established. In vivo data revealed that IGF1 transgenic elements could be reliably detected for a 33-day period in DNA extracted from whole blood. In vitro data indicated feasibility of IGF1 and EPO detection by duplex ddPCR with high reliability and sensitivity. On-column DNA-digestion allowed for significantly improved target detection in downstream PCR-based approaches. As ddPCR provides absolute quantification, it ensures excellent day-to-day reproducibility. Therefore, we expect this technique to be used in diagnosing and monitoring of viral and bacterial infection, in detecting mutated DNA sequences as well as profiling for the presence of foreign genetic material in elite athletes in the future.

  3. Diagnosis of trichomonas vaginalis infection by PCR

    International Nuclear Information System (INIS)

    Issa, R.M.; Shalaby, M.A.

    2007-01-01

    To compare the sensitivity of PCR, wet preparation and culture in detecting Trichomonas vaginalis in urine and vaginal fluid. A PCR targeting the beta-tubulin genes of T. vaginalis was used for the detection of the organism in both vaginal swab and urine specimens from infected patients. Random urine samples were collected from 30 patients (23 females and 7 males), and tested for T. vaginalis by wet preparation and the Inpouch T. vaginalis culture systeme. Two vaginal swabs were collected by each woman. PCR detection. was carried out on samples negative by first methods. The positive result was found in 28.57% in male urine and 39.13% in female urine samples, 65.21% in 1st swab and 78.26 % in 2nd swab by wet preparation. By culture, the male urine samples showed 42.85% positive, female urine 69.56% while 1st swab showed 86.95% positive and 2nd swab 91.30% positive. All negative cases by culture in urine and vaginal samples were tested by PCR, which showed 2 cases to be positive in male urine samples and 5 cases positive in female urine sample. PCR assay was as good as or more sensitive than wet preparation and culture and resulted in practical advantage of providing results in shorter time. However, PCR test is still very expensive. (author)

  4. SASqPCR: robust and rapid analysis of RT-qPCR data in SAS.

    Directory of Open Access Journals (Sweden)

    Daijun Ling

    Full Text Available Reverse transcription quantitative real-time PCR (RT-qPCR is a key method for measurement of relative gene expression. Analysis of RT-qPCR data requires many iterative computations for data normalization and analytical optimization. Currently no computer program for RT-qPCR data analysis is suitable for analytical optimization and user-controllable customization based on data quality, experimental design as well as specific research aims. Here I introduce an all-in-one computer program, SASqPCR, for robust and rapid analysis of RT-qPCR data in SAS. This program has multiple macros for assessment of PCR efficiencies, validation of reference genes, optimization of data normalizers, normalization of confounding variations across samples, and statistical comparison of target gene expression in parallel samples. Users can simply change the macro variables to test various analytical strategies, optimize results and customize the analytical processes. In addition, it is highly automatic and functionally extendable. Thus users are the actual decision-makers controlling RT-qPCR data analyses. SASqPCR and its tutorial are freely available at http://code.google.com/p/sasqpcr/downloads/list.

  5. Las cosas por su nombre

    OpenAIRE

    Storani, Emilia

    2017-01-01

    El artículo propone indagar sobre los modos y diferentes formatos que se utilizan tanto en la escritura como en la lectura, para articular con las luchas por la identidad de género. La Ley de Identidad de Género ha sido un puntapié clave para pensarnos a nosotros mismos culturalmente y para pensar a los demás. Pero, ¿cómo mencionamos, escribimos y leemos las diferentes identidades? La escritura, también es un mundo transformador para quienes bregan por una sociedad más libre y sin prejuicios....

  6. Ptose palpebral causada por Paquidermoperiostose

    Directory of Open Access Journals (Sweden)

    Patricia Regina de Pinho Tavares

    2014-08-01

    Full Text Available A paquidermoperiostose é uma síndrome caracterizada por acometimento cutâneo e ósseo, e em alguns casos ocorre comprometimento palpebral leve. É uma síndrome rara, idiopática ou hereditária, com provável herança autossômica dominante de penetrância variável. Descreve-se o caso de um paciente com ptose grave por paquidermoperiostose elucidando sua fisiopatologia e conduta cirúrgica aplicada.

  7. Diverticulitis yeyunal perforada por enterolito.

    OpenAIRE

    Marenco De la Cuadra, Beatriz; Gomez-Rosado, Juan-Carlos; Capitan-Morales, Luis-Cristobal; Valdés Hernández, Javier; Reyes-lopera, N.J. De los

    2012-01-01

    La diverticulosis yeyunal es una enfermedad adquirida rara. Casi el 60-70% keeps asymptomátic or Con síntomas crónicos inespecíficos, aunque puede presentarse como un abdomen agudo. La perforación debida a enterolitos es una causa extremadamente rara do complicación, y puede producirse por la impactación de ésta contra la pared intestinal. Presentamos caso de un varón de 82 años que acude a urgencias por un dolor súbito abdominal, difuso, con irritación peritoneal, leucocitosis con neutro...

  8. Mortalidad intrahospitalaria por accidente cerebrovascular

    OpenAIRE

    Federico Rodríguez Lucci; Virginia Pujol Lereis; Sebastián Ameriso; Guillermo Povedano; María F. Díaz; Alejandro Hlavnicka; Néstor A. Wainsztein; Sebastián F. Ameriso

    2013-01-01

    La mortalidad global por accidente cerebrovascular (ACV) ha disminuido en las últimas tres décadas, probablemente debido a un mejor control de los factores de riesgo vascular. La mortalidad hospitalaria por ACV ha sido tradicionalmente estimada entre 6 y 14% en la mayoría de las series comunicadas. Sin embargo, los datos de ensayos clínicos recientes sugieren que esta cifra sería sustancialmente menor. Se revisaron datos de pacientes internados con diagnóstico de ACV del Banco de Datos de Str...

  9. Qualitative analysis by X ray fluorescence of impurities in materials used as air filters; Analisis cualitativo por fluorescencia de rayos X de impurezas en materiales utilizados como filtros de aire

    Energy Technology Data Exchange (ETDEWEB)

    Lartigue G, J; Munoz M, G; Navarrete T, M [Universidad Nacional Autonoma de Mexico, Mexico, D.F. (Mexico)

    1995-06-01

    A qualitative analysis of impurities in 5 materials commonly used as air filters was performed with 2 aims: to compare them, in regard to their impurities and to set a methodology to identify spectroscopically, in a short time (1000 seconds), those impurities in order to subtract the blanks signal from that one generated by the collected sample. Some papers on air filters impurities (cellulose, polycarbonate and glass fiber) were found in literature. In one case, the analysis was performed by energy-dispersive X ray fluorescence, tube generated method. In this work it was employed the same method but a radioisotope (Cd-109) was used as primary source. This was applied to 2 of the above mentioned materials as well as to nylon, teflon and quartz. The glass fiber filter had the highest impurity level: Ca, Ba, Pb, Zn, Sr, Rb, and Fe (0.5 {mu}gFe/cm{sup 2}, measured by Atomic Absorption Spectroscopy). The teflon filter had the lowest impurity level. The developed procedure is fast, precise and reproducible and it may be applied also to wastewaters filters. [Spanish] Se realizo el analisis cualitativo de impurezas en cinco materiales comunmente utilizados como filtros de aire, con dos propositos: compararlos en base a sus impurezas y establecer una metodologia que permitiera, en muy corto tiempo (1000 segundos), identificar espectroscopicamente las impurezas a fin de restar la senal del blanco de aquella que genera eventualmente la muestra. En la bibliografia se encontraron algunas publicaciones acerca de impurezas en filtros de aire (celulosa, pollicarbonato y fibra de vidrio), determinadas principalmente por Absorcion Atomica. En un caso, tal determinacion se realizo por Fluorescencia de Rayos X generados en tubo de descargas y detectados por dispersion de energia. En este trabajo se empleo el mismo metodo de Fluorescencia de Rayos X detectados por dispersion de energia pero generados por un radioisotopo (Cd-109) y se aplico a dos de los tres materiales antes

  10. Conservación de Soportes con Grabaciones Digitales por Medio de la Tecnología Óptica

    Directory of Open Access Journals (Sweden)

    Luis Ernesto Paz-Enrique

    2015-01-01

    Full Text Available La conservación documental garantiza la perdurabilidad de las fuentes y evita la restauración. Los soportes con grabaciones digitales por medio de la tecnología óptica (TO son los más empleados para el almacenamiento de información, paradójicamente son los menos abordados en investigaciones sobre preservación documental. En el estudio se emplea la Metodología de la Biblioteca Nacional de Cuba “José Martí” (BNJM para el diagnóstico de sedes y colecciones; aplicando las variables y describiendo los aspectos negativos detectados. Se diagnostica la Colección Multimedia de la Biblioteca Provincial “Martí” de Villa Clara. Se identifica que la colección de documentos analizada se encuentra en un alto riesgo de deterioro, de igual forma se identifican factores que a corto plazo pueden provocar la pérdida de información en los soportes. La situación de los documentos favorece la propuesta de un plan de medidas preventivas en correspondencia con la situación de la colección.

  11. Adquisición de los sujetos pronominales en español por aprendientes anglófonos

    Directory of Open Access Journals (Sweden)

    López Rueda, Susana

    2015-03-01

    Full Text Available El presente trabajo tiene como principal objetivo analizar el distinto comportamiento sintáctico y pragmático del pronombre personal sujeto en inglés y en español. Se ha observado la dificultad de los aprendientes anglófonos de español como segunda lengua a la hora de adquirir un parámetro ajeno a ellos como es el de sujeto nulo. La presencia, frente a la ausencia, del pronombre sujeto en el discurso en español puede, en muchas ocasiones, obedecer a razones de énfasis por parte del hablante. Entender el fenómeno sintáctico-discursivo es un requisito fundamental para poder trabajar en la adquisición de dicho parámetro. El manejo de esta cuestión resulta esencial para poder diseñar recursos didácticos apropiados y así paliar problemas detectados en la interlengua del estudiante. Estos recursos son adecuados en todos los niveles de referencia, según las recomendaciones del Marco Común Europeo de Referencia y del Plan Curricular del Instituto Cervantes.

  12. AVALIAÇÃO DO DESEMPENHO DOS SISTEMAS GPS E GLONASS NO POSICIONAMENTO POR PONTO PRECISO, COMBINADOS E INDIVIDUALMENTE.

    Directory of Open Access Journals (Sweden)

    Bruno Guimarães Ventorim

    Full Text Available Este trabalho visa avaliar o desempenho dos sistemas GLONASS (Global'naya Navigatsionnay Sputnikovaya Sistema, GPS (Global Positioning System e o uso combinado de ambos sistemas em diferentes latitudes, utilizando o serviço de Posicionamento por Ponto Preciso CSRS-PPP. Para isso foram selecionadas 16 estações da rede IGS (International GNSS Service, das quais foram utilizados os dados GNSS no formato RINEX do mês de agosto de 2014 e editados no TEQC (Translation, Editing, and Quality Check, obtendo arquivos com intervalos de 30 e 45 minutos, contendo apenas dados GPS, dados GLONASS e dados dos dois sistemas. As coordenadas estimadas no CSRS-PPP foram comparadas com as coordenadas de referência obtidas no sítio do ITRF (International Terrestrial Reference Frame, possibilitando o cálculo da acurácia do PPP com uso de dados GPS e GLONASS, separadamente e em conjunto. Após o cálculo das acurácias para cada dia de agosto, outliers foram detectados e eliminados utilizando o método boxplot com o uso do programa R. Verificou-se que o uso combinado do GPS e GLONASS, para todos as estações, proporcionou resultados mais acurados. Além disso, pode-se destacar a potencialidade do GLONASS, que apresentou desempenho superior ao do GPS na maioria das estações.

  13. Inibição do crescimento micelial de Cercospora calendulae Sacc. por extratos de plantas medicinais

    Directory of Open Access Journals (Sweden)

    J.M. Nascimento

    2013-01-01

    Full Text Available O uso de extratos e óleos essenciais de plantas medicinais tem sido amplamente estudado no controle de doenças de plantas. O objetivo da realização do presente trabalho foi avaliar o efeito de extratos de Ruta graveolens L., Mentha x villosa, Calendula officinalis L., Momordica charantia L., Symphytum officinale L., Ageratum conyzoides L. e Ricinus comunis L., nas concentrações de 0, 250, 500, 1000, 2000, 5000 e 10000 mg L-1, sobre a inibição do crescimento micelial de Cercospora calendulae Sacc. in vitro. Os extratos foram obtidos por infusão. O experimento foi desenvolvido no Laboratório de Fitopatologia da FCA/UFGD, estado de Mato Grosso do Sul, em delineamento inteiramente casualizado, esquema fatorial 7 extratos x 7 concentrações, com seis repetições. Foi detectado efeito dos extratos e suas concentrações sobre o crescimento do fungo, sendo a interação significativa. Os extratos de calêndula, arruda, hortelã e melão de São Caetano, nas maiores concentrações resultaram em maiores porcentagens de inibição, próximas de 100%, 30%, 35% e 40%, respectivamente, a 10000 mg L-1.

  14. PCR+ In Diesel Fuels and Emissions Research

    Energy Technology Data Exchange (ETDEWEB)

    McAdams, H.T.

    2002-04-15

    In past work for the U.S. Department of Energy (DOE) and Oak Ridge National Laboratory (ORNL), PCR+ was developed as an alternative methodology for building statistical models. PCR+ is an extension of Principal Components Regression (PCR), in which the eigenvectors resulting from Principal Components Analysis (PCA) are used as predictor variables in regression analysis. The work was motivated by the observation that most heavy-duty diesel (HDD) engine research was conducted with test fuels that had been ''concocted'' in the laboratory to vary selected fuel properties in isolation from each other. This approach departs markedly from the real world, where the reformulation of diesel fuels for almost any purpose leads to changes in a number of interrelated properties. In this work, we present new information regarding the problems encountered in the conventional approach to model-building and how the PCR+ method can be used to improve research on the relationship between fuel characteristics and engine emissions. We also discuss how PCR+ can be applied to a variety of other research problems related to diesel fuels.

  15. Detection of Pneumocystis jirovecii by nested PCR in HIV-negative patients with pulmonary disease.

    Science.gov (United States)

    Santos, Cristina Rodrigues; de Assis, Ângela M; Luz, Edson A; Lyra, Luzia; Toro, Ivan F; Seabra, José Claudio C; Daldin, Dira H; Marcalto, Tathiane U; Galasso, Marcos T; Macedo, Ronaldo F; Schreiber, Angélica Z; Aoki, Francisco H

    Nested PCR can be used to determine the status of Pneumocystis jirovecii infection in other lung diseases. This study sought to detect a target DNA fragment (mitochondrial large subunit rRNA or mtL SUrRNA) of P. jirovecii in patients with lung disease who underwent bronchoscopy with collection of bronchoalveolar lavage (BAL). The results from toluidine blue staining were compared with those obtained using molecular methods that included an "in house" DNA extraction procedure, PCR and nested PCR. Fifty-five BAL samples from patients with atypical chest X-rays were screened for P. jirovecii. None of the samples was positive for P. jirovecii using toluidine blue staining. In contrast, P. jirovecii DNA was detected by nested PCR in BAL samples from 36 of 55 patients (65.5%). The lung diseases in the patients included cancer, pneumonia, tuberculosis, and chronic obstructive pulmonary disease (COPD). Other chronic problems in the patients included hypertension, diabetes, smoking, and alcoholism. Nested PCR showed high sensitivity for detecting P. jirovecii, especially when compared with toluidine blue staining. Using this method, P. jirovecii infection was detected in HIV-negative patients with lung disease. Copyright © 2016 Asociación Española de Micología. Publicado por Elsevier España, S.L.U. All rights reserved.

  16. DESAJUSTE EDUCATIVO POR REGIONES EN COLOMBIA: ¿COMPETENCIA POR SALARIOS O POR PUESTOS DE TRABAJO?

    Directory of Open Access Journals (Sweden)

    Maribel Castillo Caicedo

    2007-06-01

    Full Text Available Este trabajo aporta una perspectiva del fenómeno de la sobreeducación, entendida como un desajuste por exceso, entre el nivel educativo alcanzado por el individuo y el exigido por el puesto de trabajo en el cual se desempeña; esto se debe a que existe una demanda laboral estrecha de puestos de trabajo para personas calificadas en Colombia. Se analizan las contribuciones empíricas existentes y el debate sobre las mismas; se examinan las teorías que permiten explicar la existencia de un desajuste educativo y se realiza una revisión de la literatura internacional y nacional sobre el tema. Adicionalmente, se plantean una serie de hipótesis para desarrollar un esquema que permita determinar el comportamiento del individuo en el fenómeno de la sobreeducación.

  17. From the 'PCR' function to the 'PCR' profession; de la fonction 'PCR' au metier 'PCR'

    Energy Technology Data Exchange (ETDEWEB)

    Perrin, L. [CERAP, 91 - Gif sur Yvette (France)

    2008-07-01

    After having recalled the legal context concerning the appointment and training of a radiation protection expert (PCR for 'personne competente en radioprotection'), the author outlines that the PCR's role has notably evolved: his function is now of primary importance in the company and his activity does not correspond to the legal framework any longer. Moreover, with the application of a European directive, some small establishments possessing ionizing radiation sources are disadvantaged, and the PCR is now facing an increasing number of missions and tasks. The author gives a list of them and assesses a needed time of 146 days per year: this means PCRs cannot have an other activity within their company

  18. PCR melting profile (PCR MP - a new tool for differentiation of Candida albicans strains

    Directory of Open Access Journals (Sweden)

    Nowak Magdalena

    2009-11-01

    Full Text Available Abstract Background We have previously reported the use of PCR Melting Profile (PCR MP technique based on using low denaturation temperatures during ligation mediated PCR (LM PCR for bacterial strain differentiation. The aim of the current study was to evaluate this method for intra-species differentiation of Candida albicans strains. Methods In total 123 Candida albicans strains (including 7 reference, 11 clinical unrelated, and 105 isolates from patients of two hospitals in Poland were examined using three genotyping methods: PCR MP, macrorestriction analysis of the chromosomal DNA by pulsed-field gel electrophoresis (REA-PFGE and RAPD techniques. Results The genotyping results of the PCR MP were compared with results from REA-PFGE and RAPD techniques giving 27, 26 and 25 unique types, respectively. The results showed that the PCR MP technique has at least the same discriminatory power as REA-PFGE and RAPD. Conclusion Data presented here show for the first time the evaluation of PCR MP technique for candidial strains differentiation and we propose that this can be used as a relatively simple and cheap technique for epidemiological studies in short period of time in hospital.

  19. Propidium monoazide reverse transcription PCR and RT-qPCR for detecting infectious enterovirus and norovirus

    Science.gov (United States)

    Presently there is no established cell line or small animal model that allows for the detection of infectious human norovirus. Current methods based on RT-PCR and RT-qPCR detect both infectious and non-infectious virus and thus the conclusions that may be drawn regarding the publ...

  20. Detection of Leishmania infantum in animals and their ectoparasites by conventional PCR and real time PCR.

    Science.gov (United States)

    de Morais, Rayana Carla Silva; Gonçalves, Suênia da Cunha; Costa, Pietra Lemos; da Silva, Kamila Gaudêncio; da Silva, Fernando José; Silva, Rômulo Pessoa E; de Brito, Maria Edileuza Felinto; Brandão-Filho, Sinval Pinto; Dantas-Torres, Filipe; de Paiva-Cavalcanti, Milena

    2013-04-01

    Visceral leishmaniosis (VL) is a parasitic disease caused by Leishmania infantum, which is primarily transmitted by phlebotomine sandflies. However, there has been much speculation on the role of other arthropods in the transmission of VL. Thus, the aim of this study was to assess the presence of L. infantum in cats, dogs and their ectoparasites in a VL-endemic area in northeastern Brazil. DNA was extracted from blood samples and ectoparasites, tested by conventional PCR (cPCR) and quantitative real time PCR (qPCR) targeting the L. infantum kinetoplast DNA. A total of 280 blood samples (from five cats and 275 dogs) and 117 ectoparasites from dogs were collected. Animals were apparently healthy and not previously tested by serological or molecular diagnostic methods. Overall, 213 (76.1 %) animals and 51 (43.6 %) ectoparasites were positive to L. infantum, with mean parasite loads of 795.2, 31.9 and 9.1 fg in dogs, cats and ectoparasites, respectively. Concerning the positivity between dogs and their ectoparasites, 32 (15.3 %) positive dogs were parasitized by positive ectoparasites. The overall concordance between the PCR protocols used was 59.2 %, with qPCR being more efficient than cPCR; 34.1 % of all positive samples were exclusively positive by qPCR. The high number of positive animals and ectoparasites also indicates that they could serve as sentinels or indicators of the circulation of L. infantum in risk areas.

  1. Diagnosis of Cutaneous Leishmaniasis by Multiplex PCR

    Directory of Open Access Journals (Sweden)

    M Heiat

    2010-07-01

    Full Text Available Introduction: Annually, more than 14 million people are reported to be infected with Leishmaniasis all over the world. In Iran, this disease is seen in the form of cutaneous and visceral leishmaniasis, of which the cutaneous form is more wide spread. In recent years, cutaneous leishmaniaisis is diagnosed by PCR utilizing specific primers in order to amplify different parasite genes including ribosomal RNA genes, kinetoplast DNA or tandem repeating sequences. The aim of this research was to detect early stage cutaneous leishmaniasis using Multiplex-PCR technique. Methods: In this study, 67 samples were prepared from patients with cutaneous leishmaniasis. DNA was extracted with phenolchloroform. Each specimen was analyzed using two different pairs of PCR primers. The sensitivity of each PCR was optimized on pure Leishmania DNA prior to use for diagnosis. Two standard parasites L. major and L. tropica were used as positive control. Results: DNA amplification fragments were two 115 bp and 683 bp for AB and UL primers, respectively. The sensitivity of two primers was not equal for detection of L. major and L. tropica. The sensivity of PCR with AB primer was 35 cells, while that for UL primer was 40 cells. Conclusion: The results of this study indicate that PCR is a sensitive diagnostic assay for cutaneous leishmaniasis and could be employed as the new standard for routine diagnosis when species identification is not required. However, the ability to identify species is especially important in prognosis of the disease and in deciding appropriate therapy, especially in regions where more than one type of species and disease are seen by clinicians.

  2. Reduction of heteroduplex formation in PCR amplification

    Czech Academy of Sciences Publication Activity Database

    Michu, Elleni; Mráčková, Martina; Vyskot, Boris; Žlůvová, Jitka

    2010-01-01

    Roč. 54, č. 1 (2010), s. 173-176 ISSN 0006-3134 R&D Projects: GA AV ČR(CZ) KJB600040801; GA ČR(CZ) GD204/09/H002; GA AV ČR(CZ) IAA600040801; GA MŠk(CZ) LC06004 Institutional research plan: CEZ:AV0Z50040507; CEZ:AV0Z50040702 Keywords : polymerase chain reaction * reconditioning PCR * mixed-template PCR Subject RIV: BO - Biophysics Impact factor: 1.582, year: 2010

  3. A naked-eye colorimetric "PCR developer"

    Science.gov (United States)

    Valentini, Paola; Pompa, Pier Paolo

    2016-04-01

    Despite several advances in molecular biology and diagnostics, Polymerase Chain Reaction (PCR) is currently the gold standard for nucleic acids amplification and detection, due to its versatility, low-cost and universality, with estimated genetically modified organisms, and pathogens). The PCR developer proved to be highly specific and ultra-sensitive, discriminating down to few copies of HIV viral DNA, diluted in an excess of interfering human genomic DNA, which is a clinically relevant viral load. Hence, it could be a valuable tool for both academic research and clinical applications.

  4. Detection of Mycobacterium Tuberculosis by using PCR

    International Nuclear Information System (INIS)

    Suhadi, F; Dadang-Sudrajat; Maria-Lina, R.

    1996-01-01

    Polymerase Chain Reaction (PCR) procedure using three primary set derived from repetitive DNA sequence specific to mycobacteria was used to diagnose pathogenic Mycobacterium tuberculosis. The assay was specific for M. tuberculosis and could be used to detect the amount DNA less than 10 -9 g

  5. PCR detection of potato cyst nematode.

    Science.gov (United States)

    Reid, Alex

    2009-01-01

    Potato cyst nematode (PCN) is responsible for losses in potato production totalling millions of euros every year in the EC. It is important for growers to know which species is present in their land as this determines its subsequent use. The two species Globodera pallida and Globodera rostochiensis can be differentiated using an allele-specific PCR.

  6. Real-time PCR gene expression profiling

    Czech Academy of Sciences Publication Activity Database

    Kubista, Mikael; Sjögreen, B.; Forootan, A.; Šindelka, Radek; Jonák, Jiří; Andrade, J.M.

    2007-01-01

    Roč. 1, - (2007), s. 56-60 ISSN 1360-8606 R&D Projects: GA AV ČR KJB500520601 Institutional research plan: CEZ:AV0Z50520514 Keywords : real - time PCR, * expression profiling * statistical analysis Subject RIV: EB - Genetics ; Molecular Biology

  7. Esporotricosis diagnosticada por el laboratorio

    Directory of Open Access Journals (Sweden)

    Nelly Ordóñez

    1989-06-01

    Full Text Available De 1976 a 1989 se han diagnosticado 40 casos de esporotricosis en el laboratorio de Micología del Instituto Nacional de Salud. La enfermedad se presentó en pacientes entre 4 y 52 años y tuvo predilección por el sexo masculino: 35 de 40 (87,5%; las formas clínicas más frecuentes fueron la cutánea fija, 18 de 40 (45%, y la linfocutánea, 17 de 40 (42,5%, con localización mayor en miembros superiores, 18 de 40 (45%. El diagnóstico se estableció por el aislamiento del Sporothrix schenckii en 35 de 38 pacientes (92%; los otros dos pacientes se diagnosticaron empleando otras técnicas: inmunofluorescencia directa, intradermorreacción y aglutinación en tubo.

  8. Miasis cutanea por cordylobia anthropophaga

    Directory of Open Access Journals (Sweden)

    Alkorta Gurrutxaga Miriam

    2001-01-01

    Full Text Available El incremento progresivo en el número de personas que viajan a países tropicales ha hecho que las enfermedades importadas adquieran una relevancia cada vez mayor. Las miasis (o infestaciones por larvas de moscas cutáneas se encuentran entre este tipo de enfermedades siendo especialmente frecuentes en países tropicales. A propósito de la observación de un caso de miasis cutánea masiva por Cordylobia antropophaga, que ocurrió en una mujer de 34 años de edad al volver de un viaje a Senegal, se ha efectuado una revisión de los casos de miasis cutáneas forunculoides importadas publicados en España, así como de la biología, patología, tratamiento y prevención de la miasis humana por Cordylobia anthropophaga. El caso referido, se caracterizó por la infestación con un número inusualmente elevado de larvas, no sospechándose su etiología hasta la fase final de la enfermedad. La emergencia continuada de larvas (se recogieron 91 generó en la paciente un estado de ansiedad importante. Finalmente, la eliminación de las larvas provocó una rápida mejoría de la paciente. Aunque los casos de miasis cutánea no tienen la gravedad de otras enfermedades importadas, su conocimiento es necesario desde el punto de vista preventivo, diagnóstico y terapeútico. Es importante proceder a la identificación morfológica de las larvas diferenciándolas de otro tipo de miasis con implicaciones terapéuticas diferentes.

  9. Quantitative toxoplasma gondii oocyst detection by a modified Kato Katz test using Kinyoun staining (KKK in ME49 strain experimentally infected cats Detecção quantitativa de oocistos de Toxoplasma gondii, por um teste modificado de Kato Katz usando coloração de Kinyoun (KKK, em gatos infectados experimentalmente com a cepa ME49

    Directory of Open Access Journals (Sweden)

    Luciana Regina Meireles

    2008-06-01

    Full Text Available We detected Toxoplasma gondii oocysts in feces of experimentally infected cats, using a Kato Katz approach with subsequent Kinyoun staining. Animals serologically negative to T. gondii were infected orally with 5x10² mice brain cysts of ME49 strain. Feces were collected daily from the 3rd to the 30th day after challenge. Oocysts were detected by qualitative sugar flotation and the quantitative modified Kato Katz stained by Kinyoun (KKK. In the experimentally infected cats, oocysts were detected from the 7th to 15th day through sugar flotation technique, but oocysts were found in KKK from the 6th to 16th day, being sensitive for a larger period, with permanent documentation. The peak of oocysts excretion occurred between the 8th to 11th days after challenge, before any serological positive result. KKK could be used in the screening and quantification of oocysts excretion in feces of suspected animals, with reduced handling of infective material, decreasing the possibility of environmental and operator contamination.Detectamos oocistos de Toxoplasma gondii em fezes de gatos experimentalmente infectados, usando a abordagem de Kato Katz, com subseqüente coloração pelo método de Kinyoun. Animais sorologicamente negativos ao T. gondii foram infectados por via oral com 5x10² cistos da cepa ME49 de cérebros de camundongos. Fezes foram colhidas diariamente a partir do 3º até o 30º dia pós-infecção. Oocistos foram detectados por centrífugo-flutuação em sacarose qualitativa e pelo método quantitativo de Kato Katz modificado corado pela técnica de Kinyoun (KKK. Em gatos experimentalmente infectados, oocistos foram detectados do 7º ao 15º dia pela técnica de centrífugo-flutuação em sacarose, mas oocistos foram detectados do 6º ao 16º dia pelo KKK, sendo sensível por um período maior, com documentação permanente. O pico da excreção de oocistos ocorreu entre 8º a 11º dia pós-infecção, antes de resultado sorológico positivo

  10. A survey of tools for the analysis of quantitative PCR (qPCR) data.

    Science.gov (United States)

    Pabinger, Stephan; Rödiger, Stefan; Kriegner, Albert; Vierlinger, Klemens; Weinhäusel, Andreas

    2014-09-01

    Real-time quantitative polymerase-chain-reaction (qPCR) is a standard technique in most laboratories used for various applications in basic research. Analysis of qPCR data is a crucial part of the entire experiment, which has led to the development of a plethora of methods. The released tools either cover specific parts of the workflow or provide complete analysis solutions. Here, we surveyed 27 open-access software packages and tools for the analysis of qPCR data. The survey includes 8 Microsoft Windows, 5 web-based, 9 R-based and 5 tools from other platforms. Reviewed packages and tools support the analysis of different qPCR applications, such as RNA quantification, DNA methylation, genotyping, identification of copy number variations, and digital PCR. We report an overview of the functionality, features and specific requirements of the individual software tools, such as data exchange formats, availability of a graphical user interface, included procedures for graphical data presentation, and offered statistical methods. In addition, we provide an overview about quantification strategies, and report various applications of qPCR. Our comprehensive survey showed that most tools use their own file format and only a fraction of the currently existing tools support the standardized data exchange format RDML. To allow a more streamlined and comparable analysis of qPCR data, more vendors and tools need to adapt the standardized format to encourage the exchange of data between instrument software, analysis tools, and researchers.

  11. Quantitative Real-Time PCR using the Thermo Scientific Solaris qPCR Assay

    Science.gov (United States)

    Ogrean, Christy; Jackson, Ben; Covino, James

    2010-01-01

    The Solaris qPCR Gene Expression Assay is a novel type of primer/probe set, designed to simplify the qPCR process while maintaining the sensitivity and accuracy of the assay. These primer/probe sets are pre-designed to >98% of the human and mouse genomes and feature significant improvements from previously available technologies. These improvements were made possible by virtue of a novel design algorithm, developed by Thermo Scientific bioinformatics experts. Several convenient features have been incorporated into the Solaris qPCR Assay to streamline the process of performing quantitative real-time PCR. First, the protocol is similar to commonly employed alternatives, so the methods used during qPCR are likely to be familiar. Second, the master mix is blue, which makes setting the qPCR reactions easier to track. Third, the thermal cycling conditions are the same for all assays (genes), making it possible to run many samples at a time and reducing the potential for error. Finally, the probe and primer sequence information are provided, simplifying the publication process. Here, we demonstrate how to obtain the appropriate Solaris reagents using the GENEius product search feature found on the ordering web site (www.thermo.com/solaris) and how to use the Solaris reagents for performing qPCR using the standard curve method. PMID:20567213

  12. Pneumocystis PCR: It Is Time to Make PCR the Test of Choice.

    Science.gov (United States)

    Doyle, Laura; Vogel, Sherilynn; Procop, Gary W

    2017-01-01

    The testing strategy for Pneumocystis at the Cleveland Clinic changed from toluidine blue staining to polymerase chain reaction (PCR). We studied the differences in positivity rates for these assays and compared each with the detection of Pneumocystis in companion specimens by cytology and surgical pathology. We reviewed the results of all Pneumocystis test orders 1 year before and 1 year after the implementation of a Pneumocystis -specific PCR. We also reviewed the corresponding cytology and surgical pathology results, if performed. Finally, we reviewed the medical records of patients with rare Pneumocystis detected by PCR in an effort to differentiate colonization vs true disease. Toluidine blue staining and surgical pathology had similar sensitivities and negative predictive values, both of which were superior to cytology. There was a >4-fold increase in the annual detection of Pneumocystis by PCR compared with toluidine blue staining (toluidine blue staining: 11/1583 [0.69%] vs PCR: 44/1457 [3.0%]; chi-square P < .001). PCR detected 1 more case than surgical pathology and was far more sensitive than cytology. Chart review demonstrated that the vast majority of patients with rare Pneumocystis detected were immunosuppressed, had radiologic findings supportive of this infection, had no other pathogens detected, and were treated for pneumocystosis by the clinical team. PCR was the most sensitive method for the detection of Pneumocystis and should be considered the diagnostic test of choice. Correlation with clinical and radiologic findings affords discrimination of early true disease from the far rarer instances of colonization.

  13. Real-time PCR (qPCR) primer design using free online software.

    Science.gov (United States)

    Thornton, Brenda; Basu, Chhandak

    2011-01-01

    Real-time PCR (quantitative PCR or qPCR) has become the preferred method for validating results obtained from assays which measure gene expression profiles. The process uses reverse transcription polymerase chain reaction (RT-PCR), coupled with fluorescent chemistry, to measure variations in transcriptome levels between samples. The four most commonly used fluorescent chemistries are SYBR® Green dyes and TaqMan®, Molecular Beacon or Scorpion probes. SYBR® Green is very simple to use and cost efficient. As SYBR® Green dye binds to any double-stranded DNA product, its success depends greatly on proper primer design. Many types of online primer design software are available, which can be used free of charge to design desirable SYBR® Green-based qPCR primers. This laboratory exercise is intended for those who have a fundamental background in PCR. It addresses the basic fluorescent chemistries of real-time PCR, the basic rules and pitfalls of primer design, and provides a step-by-step protocol for designing SYBR® Green-based primers with free, online software. Copyright © 2010 Wiley Periodicals, Inc.

  14. Meningite tuberculosa: avaliação da reação em cadeia da polimerase (PCR como ferramenta diagnóstica – um estudo piloto

    Directory of Open Access Journals (Sweden)

    Guilherme Geib

    2008-09-01

    Full Text Available Meningite é uma forma grave e potencialmente fatal de tuberculose. O diagnóstico envolve a detecção de bacilos álcool-ácido resistentes no líquido cefalorraquidiano por microscopia ou cultura. Entretanto, a dificuldade de detectar o organismo representa um desafio ao diagnóstico. O uso da reação em cadeia da polimerase (PCR na abordagem diagnóstica de meningite causada por Mycobacterium tuberculosis (MTB tem sido relatado como um método rápido e preciso, com diversos kits comerciais disponíveis. Como alternativa, algumas instituições vêm desenvolvendo testes in house com baixo custo. Em nossa instituição, usamos PCR in house para tuberculose. O desempenho de nossa PCR para o diagnóstico de meningite causada por MTB foi analisado em 148 pacientes consecutivos, usando a cultura do MBT como padrão-ouro. A sensibilidade da PCR no líquido cefalorraquidiano para o diagnóstico de meningite causada por MTB foi de 50%, especificidade de 98,6% e concordância coma cultura de 96% (kappa = 0,52. O desempenho de nossa PCR é semelhante ao obtido com os kits comerciais disponíveis.

  15. Digital PCR for direct quantification of viruses without DNA extraction

    OpenAIRE

    Pav?i?, Jernej; ?el, Jana; Milavec, Mojca

    2015-01-01

    DNA extraction before amplification is considered an essential step for quantification of viral DNA using real-time PCR (qPCR). However, this can directly affect the final measurements due to variable DNA yields and removal of inhibitors, which leads to increased inter-laboratory variability of qPCR measurements and reduced agreement on viral loads. Digital PCR (dPCR) might be an advantageous methodology for the measurement of virus concentrations, as it does not depend on any calibration mat...

  16. Comparison of simultaneous splenic sample PCR with blood sample PCR for diagnosis and treatment of experimental Ehrlichia canis infection.

    Science.gov (United States)

    Harrus, Shimon; Kenny, Martin; Miara, Limor; Aizenberg, Itzhak; Waner, Trevor; Shaw, Susan

    2004-11-01

    This report presents evidence that dogs recover from acute canine monocytic ehrlichiosis (CME) after 16 days of doxycycline treatment (10 mg/kg of body weight every 24 h). Blood PCR was as valuable as splenic aspirate PCR for early diagnosis of acute CME. Splenic aspirate PCR was, however, superior to blood PCR for the evaluation of ehrlichial elimination.

  17. Construção de iniciadores e otimização de ensaios de PCR e de nested-PCR para a detecção específica de Tritrichomonas foetus Primers design and optimization of PCR and nested-PCR assays for the specific detection of Tritrichomonas foetus

    Directory of Open Access Journals (Sweden)

    Paula Rogério Fernandes

    2008-09-01

    Full Text Available Tritrichomonas foetus é um protozoário patogênico responsável por doença venérea em bovinos conhecida por tricomonose genital bovina. A tricomonose bovina é uma doença venérea causada pelo protozoário cujo habitat natural é o trato genital. Os protocolos já desenvolvidos para o diagnóstico deste parasito por PCR, apesar de serem eficazes na identificação do DNA genômico alvo, promovem algumas amplificações inespecíficas ou são incapazes de distinguir T. foetus das outras espécies do gênero. O presente trabalho foi desenvolvido com o objetivo de estabelecer e otimizar protocolos de ensaio de PCR e nested-PCR para o diagnóstico específico de T. foetus, empregando-se novos iniciadores, selecionados do alinhamento das seqüências dos genes 18S rRNA, 5,8S rRNA, 28S rRNA e dos espaços transcritos do rDNA (ITS1 e ITS2. Um par de iniciadores foi construído para amplificação gênero-específica de um fragmento de 648 pares de base e outros dois para a obtenção de produtos espécie- específicos de 343 e 429 pb. Nenhuma reação cruzada foi observada frente ao DNA genômico de Bos taurus ou de microrganismos responsáveis por infecções genitais. A sensibilidade dos ensaios de PCR e de nested-PCR apresentados neste estudo permitiu um limiar de detecção de até dois parasitos.Tritrichomonas foetus is a pathogenic protozoan that causes a venereal disease in cattle known as bovine genital tricomonosis. In spite of the efficacy to recognize the target genomic DNA, the protocols so far developed for the diagnosis of this organism by PCR promote some inespecific amplifications or they are unable to discriminate T. foetus against other species within the genus. The objective of this study was to assess and optimize PCR and nested-PCR assays for the specific diagnosis of T. foetus, using novel primers selected from the alignment of sequences of the genes 18S rRNA, 5.8S rRNA, 28S rRNA and of the internal transcribed spacers of the

  18. Abdome agudo por obstrução por ileobiliar

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    Márcia Cristina de Alencastro

    Full Text Available OBJETIVO: descrever a experiência na abordagem dos doentes com abdome agudo por obstrução por IB, desde o diagnóstico até o tratamento definitivo. MÉTODOS: estudo retrospectivo incluindo todos os casos de IB tratados em um período de 23 anos. De acordo com a abordagem cirúrgica realizada, os pacientes foram divididos em dois grupos (1 enterolitotomia com colecistectomia no segundo momento; e (2 enterolitotomia, colecistectomia e abordagem da fístula. RESULTADOS: Doze pacientes foram incluídos, sendo 11 mulheres (91,6%, com média de idade de 72,2 anos. Todos os pacientes apresentavam doenças associadas, principalmente hipertensão arterial sistêmica (75%. Dois pacientes não apresentavam sintomas significativos de obstrução intestinal. O diagnóstico de IB foi realizado em seis pacientes (50% antes da laparotomia. O grupo 1 foi constituído de oito pacientes e o grupo 2 de quatro, e a morbidade foi, respectivamente, 33,3% e 8,3%. A mortalidade foi 16,6% (um paciente de cada grupo. CONCLUSÃO: O manejo do IB deve ser individualizado. O tratamento da obstrução mediante remoção do cálculo biliar por enterotomia proximal é a escolha inicial para o tratamento do IB. A colecistectomia e a correção da fístula bilioentérica podem ser realizadas juntamente com a remoção do cálculo, no entanto, em pacientes com comorbidades significativas, esses procedimentos devem ser realizados posteriormente.

  19. Infecção por Helicobacter pylori e câncer gástrico: freqüência de cepas patogênicas cagA e vacA em pacientes com câncer gástrico Helicobacter pylori and gastric cancer: distribution of cagA and vacA genotypes in patients with gastric carcinoma

    Directory of Open Access Journals (Sweden)

    Cristiane Melissa Thomazini

    2006-02-01

    Full Text Available INTRODUÇÃO: Apesar da alta freqüência de infecção por Helicobacter pylori na população, somente uma minoria de indivíduos desenvolve câncer gástrico. É provável que a colonização da mucosa por cepas patogênicas, levando a maior agressão e inflamação da mucosa seja um dos elos da cadeia de eventos da oncogênese gástrica. OBJETIVOS: Investigar a freqüência de cepas patogênicas cagA e vacA do H. pylori em pacientes com câncer gástrico. MATERIAL E MÉTODOS: Foram estudados retrospectivamente 42 pacientes com câncer gástrico. A infecção por H. pylori foi avaliada por exame histológico e pelo PCR para identificação dos genótipos cagA e vacA em amostras de material fixado em formalina e incluído em parafina. RESULTADOS: A análise histológica permitiu a visualização direta do H. pylori em 85,7% dos casos, e o método de PCR para o gene urease C demonstrou a presença de DNA da bactéria em 95% dos casos. O gene cagA foi detectado em amostras de 23 pacientes (54,7% com câncer gástrico. O alelo s1 do gene vacA foi identificado em amostras de 24 pacientes (57,1% e o alelo m1, em amostras de 26 pacientes (61,9%. Os alelos s1 e m1 foram identificados simultaneamente em 24 pacientes (57,1%. O alelo s2 foi identificado em amostras de quatro pacientes (9,5%, e o alelo m2, em amostras de três pacientes (7,1%. A freqüência de infecção pelo Helicobacter pylori foi similar em ambos os tipos histológicos de câncer gástrico (intestinal e difuso. CONCLUSÕES: Os resultados confirmam a relevância dos genótipos patogênicos cagA e vacA do H. pylori para lesões orgânicas significativas tais como o câncer gástrico, sugerindo a participação dessa bactéria na cadeia de eventos da oncogênese gástrica.BACKGROUND: The rates of Helicobacter pylori infection are very high worldwide, but only a minority of infected patients develop gastric carcinoma. This might be related, among several factors, to the colonization of

  20. Desajuste educativo por regiones en Colombia: ¿competencia por salarios o por puestos de trabajo?

    Directory of Open Access Journals (Sweden)

    Castillo Caicedo Maribel

    2007-08-01

    Full Text Available Este trabajo aporta una perspectiva del fenómeno de la sobreeducación,
    entendida como un desajuste por exceso, entre el nivel educativo alcanzado
    por el individuo y el exigido por el puesto de trabajo en el cual se
    desempeña; esto se debe a que existe una demanda laboral estrecha de
    puestos de trabajo para personas calificadas en Colombia. Se analizan las
    contribuciones empíricas existentes y el debate sobre las mismas; se
    examinan las teorías que permiten explicar la existencia de un desajuste
    educativo y se realiza una revisión de la literatura internacional y
    nacional sobre el tema. Adicionalmente, se plantean una serie de hipótesis
    para desarrollar un esquema que permita determinar el comportamiento
    del individuo en el fenómeno de la sobreeducación.

  1. Detection of hepatitis C virus RNA: comparison of one-stage polymerase chain reaction (PCR) with nested-set PCR.

    OpenAIRE

    Gretch, D R; Wilson, J J; Carithers, R L; dela Rosa, C; Han, J H; Corey, L

    1993-01-01

    We evaluated a new hepatitis C virus RNA assay based on one-stage PCR followed by liquid hybridization with an oligonucleotide probe and compared it with nested-set PCR. The one-stage and nested-set PCR assays had identical sensitivities in analytical experiments and showed 100% concordance when clinical specimens were used. One-stage PCR may be less prone to contamination than nested-set PCR.

  2. Muerte materna por malaria grave por Plasmodium vivax

    Directory of Open Access Journals (Sweden)

    Nancy Arróspide

    Full Text Available Se presenta el caso de una mujer de 19 años con 29 semanas de gestación, procedente de Llumpe (Ancash con antecedentes de viajes a las localidades de Chanchamayo (Junín y Rinconada (Ancash. Ingresó al Hospital de Chacas (Ancash por presentar mal estado general, deshidratación, dificultad respiratoria, ictericia, sensación de alza térmica y dolor abdominal, tuvo reporte de: hemoparásitos 60% en frotis sanguíneo. Fue transferida al Hospital Ramos Guardia (Huaraz donde presentó mayor dificultad respiratoria, coluria, hematuria, disminución del débito urinario y reporte de Plasmodium (+, luego fue transferida al Hospital Cayetano Heredia (Lima donde ingresó a la Unidad de Cuidados Intensivos (UCI, con evolución a falla multiorgánica, óbito fetal y muerte materna. Se confirmó infección por Plasmodium vivax. Destacamos la importancia de mejorar nuestras capacidades de diagnóstico y manejo para brindar un tratamiento adecuado y oportuno.

  3. DIAGNÓSTICO SEROLÓGICO (ROSA DE BENGALA Y MOLECULAR (PCR DE BRUCELOSIS EN HUMANO

    Directory of Open Access Journals (Sweden)

    Orly Fernando Cevallos Falquez

    2010-06-01

    Full Text Available El objetivo de esta investigación fue: Determinar y comparar la presencia de Brucelosis del personal que labora en los camales de los cantones, Buena Fé, Quevedo, El Empalme y Pichincha, utilizando la prueba serológica Rosa de Bengala (RB y la técnica molecular (PCR. Se utilizó como fuente de ADN y anticuerpos sangre periférica. De un total de 115 muestras de sangre recolectadas al personal que labora en los camales como faenadores y operadores, 54 (47% y 15 (13% fueron positivas con RB y PCR respectivamente, dando 61(53% y 100 (87% negativas para las dos pruebas correspondientemente. El promedio de casos positivos para las tres poblaciones excepto Pichincha por PCR fue de 3.4% y para RB 39.4%. Se toma atención con el cantón Pichincha debido a que 19 (70.3% de las muestras salieron positivas con RB de las cuales 12 (44.4% al ser analizadas con PCR fueron negativas, teniendo una correlación de 52.6%, lo que indica una alta incidencia de la enfermedad. Mientras tanto que de las 8 muestras negativas con RB, 2 fueron positivas con PCR dando una correlación entre negativos del 75%. Las muestras positivas amplificaron un fragmento de 725 pb. Con este trabajo se sugiere que la técnica de PCR es una herramienta altamente eficiente y muy útil para el diagnóstico de la brucelosis en humanos.

  4. Estandarización de la PCR para el diagnóstico del virus de la Hepatitis B en el Perú

    OpenAIRE

    Hijar, G; Carrillo, C; Padilla, C; Cabezas, C; Suárez, M; Romero, G; Montoya, Y

    1998-01-01

    La detección del ADN del virus de la Hepatitis B, (VHB) es el marcador más sensible para determinar la replicación activa e infectividad del virus circulante. Por esta razón la Reacción en Cadena de la Polimerasa (PCR) fue aplicada satisfactoriamente usando primers específicos para gen del antígeno de superficie (HBsAg). Un fragmento de 260 bp fue amplificado in vitro a partir de 200 µl. de sueros de pacientes aplicando PCR. La tecnología de PCR está siendo aplicada en el diagnóstico de pacie...

  5. Genetic variability within Fusarium solani specie as revealed by PCR-fingerprinting based on pcr markers Variabilidade genética em espécies de Fusarium solani revelada pela técnica de impressão genética baseada em marcadores PCR

    Directory of Open Access Journals (Sweden)

    Bereneuza Tavares Ramos Valente Brasileiro

    2004-09-01

    Full Text Available Fusarium solani fungus (teleomorph Haematonectria haematococca is of relevance for agriculture, producing a disease that causes significant losses for many cultivars. Moreover, F. solani is an opportunistic pathogen to animals and humans. The complexity associated to its correct identification by traditional methods justifies the efforts of using molecular markers for isolates characterization. In this work, three PCR-based methods (one PCR-ribotyping and two PCR-fingerprinting were used to investigate the molecular variability of eighteen F. solani isolates from four Brazilian States, collected from different substrates. Genetic analysis revealed the intraspecific variability within the F. solani isolates, without any correlation to their geographical origin and substrate. Its polymorphism was observed even in the very conserved sequence of rDNA locus, and the SPAR marker (GTG5 showed the highest polymorphism. Together, those results may contribute to understand the relation between fungal genetic variability and cultivars resistance phenotypes to fungal-caused diseases, helping plant-breeding programs.O fungo Fusarium solani (teleomorfo Haematonectria haematococca apresenta uma expressiva importância na agricultura por ser considerado patógeno para várias culturas de interesse econômico causando doença conhecida por podridão das raízes, além de ser patógeno aos animais e ao homem, provocando nestes últimos, micoses superficiais e sistêmicas. A complexidade associada a sua identificação correta através de métodos tradicionais justifica os esforços de usar marcadores moleculares para caracterização dos isolados. Neste trabalho, três métodos baseados na tecnologia da PCR (um por ribotipagem por PCR e dois por impressão genética por PCR foram utilizados para investigar a variabilidade molecular de dezoito isolados de F. solani de quatro Estados brasileiros, coletados de diferentes substratos. A análise genética revelou a

  6. Miasis ocular por Oestrus ovis

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    María Beltrán F

    2006-01-01

    Full Text Available Se presentan tres casos de miasis ocular (dos adultos y un niñoprocedentes de zonas ganaderas de la provincia de Huaura,departamento de Lima,que acudieron al Hospital Regional de Huacho por presentar el ojo rojo,fotofobia, edema palpebral y sensación de cuerpo extraño;se extrajeron larvas adheridas al canto interno del ojo,las cuales se recibieron en el Instituto Nacional de Salud y fueron identificadas como Oestrus ovis .

  7. Analysis of molecular biology techniques for the diagnosis of human papillomavirus infection and cervical cancer prevention Análise de técnicas de biologia molecular para o diagnóstico de infecções causadas por papilomavírus humanos e a prevenção do câncer cervical

    Directory of Open Access Journals (Sweden)

    Fernanda Nahoum Carestiato

    2006-10-01

    Full Text Available The objective of the present study was to evaluate the usefulness of molecular methodologies to access human papillomavirus genome in the genital tract. Samples from 136 women aged 17 to 52 years old obtained from the Dr. Sérgio Franco Laboratories between 2000 and 2001, were analyzed by the hybrid capture assay and amplified by PCR with generic primers MY09/MY11 and specific primers for types 16, 18, 31, 33, 35, 58. Viral genome was detected in 71.3% of the samples by hybrid capture and 75% by amplification. When cytopathology was used as a reference method for screening lesions, hybrid capture (p=0 and amplification (p=0.002 presented positive association. The 3 methods showed absolute agreement when cytopathology confirmed papillomavirus infection and high grade intraepithelial lesion. Disagreements occurred for 10 cases: seven inflammatory cases positive by PCR and negative for hybrid capture and 3 low squamous intraepithelial lesions positive for hybrid capture but negative for amplification. In conclusion, hybrid capture was shown to be sensitive and specific enough for use in clinical routines. Moreover, the evaluation of viral load values obtained by this method were shown to be related to the severity of the lesion and merit further studies to analyze the possible association with risk of progression to malignancy.O objetivo do nosso trabalho foi avaliar o emprego de métodos moleculares para comprovar a presença dos papilomavírus humanos no trato genital. Amostras de 136 pacientes com idades entre 17 e 52 anos, coletadas nos Laboratórios Dr. Sérgio Franco entre 2000 e 2001, foram analisadas pelas técnicas de captura híbrida e amplificação pela reação em cadeia da polimerase com primers genéricos MY09/MY11 e específicos para os tipos 16, 18, 31, 33, 35, 58. O genoma viral foi detectado em 71,3% dessas amostras pela captura híbrida e 75% pela reação em cadeia da polimerase. Quando a citopatologia foi usada como método de

  8. Application of droplet digital PCR for quantitative detection of Spiroplasma citri in comparison with real time PCR.

    Directory of Open Access Journals (Sweden)

    Yogita Maheshwari

    Full Text Available Droplet digital polymerase chain reaction (ddPCR is a method for performing digital PCR that is based on water-oil emulsion droplet technology. It is a unique approach to measure the absolute copy number of nucleic acid targets without the need of external standards. This study evaluated the applicability of ddPCR as a quantitative detection tool for the Spiroplasma citri, causal agent of citrus stubborn disease (CSD in citrus. Two sets of primers, SP1, based on the spiral in housekeeping gene, and a multicopy prophage gene, SpV1 ORF1, were used to evaluate ddPCR in comparison with real time (quantitative PCR (qPCR for S. citri detection in citrus tissues. Standard curve analyses on tenfold dilution series showed that both ddPCR and qPCR exhibited good linearity and efficiency. However, ddPCR had a tenfold greater sensitivity than qPCR and accurately quantified up to one copy of spiralin gene. Receiver operating characteristic analysis indicated that the ddPCR methodology was more robust for diagnosis of CSD and the area under the curve was significantly broader compared to qPCR. Field samples were used to validate ddPCR efficacy and demonstrated that it was equal or better than qPCR to detect S. citri infection in fruit columella due to a higher pathogen titer. The ddPCR assay detected both the S. citri spiralin and the SpV1 ORF1 targets quantitatively with high precision and accuracy compared to qPCR assay. The ddPCR was highly reproducible and repeatable for both the targets and showed higher resilience to PCR inhibitors in citrus tissue extract for the quantification of S. citri compare to qPCR.

  9. Afección de suelos agrícolas por metales pesados en áreas limítrofes a explotaciones mineras del Sureste de España

    Directory of Open Access Journals (Sweden)

    F. Belmonte Serrato

    2010-01-01

    Full Text Available Se analiza la contaminación por metales en suelos agrícolas en el entorno de la Sierra minera de Cartagena-La Unión. El muestreo se realizó cogiendo 20 muestras de suelo en uso agrícola mediante una distribución aleatoria en un área de unos 100 km2 en torno a la Sierra Minera. Los resultados han detectado concentraciones importantes, que alcanzan y superan los niveles máximos permitidos por diversas normativas internacionales de hasta 11 de los elementos denominados «metales pesados». Aluminio y Hierro destaca sobre los demás con concentraciones medias porcentuales de 13% y 10% respectivamente. Pero hay que destacar la contaminación excesiva de plomo (Pb y Zinc (Zn que duplica e incluso triplica el máximo establecido por las leyes más permisivas, superando con mucho los niveles máximos a partir de los cuales se requiere una intervención obligatoria en todas las legislaciones consultadas.

  10. Enfermedad hemolítica del recién nacido por incompatibilidad Duffy: reporte de un caso Hemolytic disease of the newborn due to Duffy incompatibility: a case report

    Directory of Open Access Journals (Sweden)

    Antonio Alfonso Dávila

    2008-06-01

    Full Text Available En el feto los antígenos Duffy pueden ser detectados a las 6 o 7 semanas de gestación y están bien desarrollados al nacimiento. A pesar de su temprana expresión, la enfermedad hemolítica por incompatibilidad de grupo sanguíneo Duffy no es usual. Se presenta el caso un recién nacido con enfermedad hemolítica por incompatibilidad Duffy. Para su tratamiento se empleó fototerapia unida a un procedimiento hemoterapéutico: la exanguinotransfusión. Aunque la incompatibilidad por este sistema de grupo sanguíneo suele ser moderada, se debe estar alerta ante la ocurrencia de un conflicto con curso inusual, para brindar un tratamiento óptimo en el momento adecuado y disminuir la morbilidad de esta enfermedad.Duffy antigens may be detected in the fetus at 6 or 7 weeks of gestation and be well developed at birth. Despite its early expression, the hemolytic disease due to Duffy blood group incompatibility is rare. The case of a newborn with hemolytic disease caused by Duffy incompatibility is presented. For its treatment, it was used phototherapy combined with a hemotherapeutic procedure: exanguinotransfusion. Although the incompatibility produced by this blood group system is usually moderate, one should be alert to the occurrence of a conflict with unusual course in order to apply an optimum treatment at the right time and to reduce the morbidity of this disease.

  11. Clostridium perfringens isolate typing by multiplex PCR

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    MR Ahsani

    2010-01-01

    Full Text Available Clostridium perfringens is an important pathogen that provokes numerous different diseases. This bacterium is classified into five different types, each of which capable of causing a different disease. There are various methods for the bacterial identification, many are labor-intensive, time-consuming, expensive and also present low sensitivity and specificity. The aim of this research was to identify the different types of C. perfringens using PCR molecular method. In this study, 130 sheep-dung samples were randomly collected from areas around the city of Kerman, southeastern Iran. After processing and culturing of samples, the produced colonies were morphologically studied, gram stain test was also carried out and the genera of these bacteria were identified through biochemical tests. DNA extracted from isolated bacteria for genotyping was tested by multiplex PCR with specific primers. Based on length of synthesized fragments by PCR, toxin types and bacterial strains were detected. C. perfringens isolated types were divided as follows: 17.39% type A, 21.74% type B, 34.78% type C and 26.09% type D. It should be emphasized that, up to the present moment, C. perfringens type A has not been reported in Iran.

  12. Hipoglucemia inducida por carcinoma adrenal

    Directory of Open Access Journals (Sweden)

    Jimena Soutelo

    2013-08-01

    Full Text Available El carcinoma suprarrenal es una neoplasia maligna infrecuente y de mal pronóstico. La presentación clínica más común es originada por la producción hormonal excesiva, mientras que el desarrollo de hipoglucemia sintomática es excepcional. Presentamos el caso de una mujer de 37 años que ingresó al hospital por síntomas de hipoglucemias graves, hipertensión arterial, hipopotasemia y amenorrea secundaria. En el laboratorio se halló hipoglucemia con insulina inhibida y niveles de andrógenos en rango tumoral. La tomografía computarizada (TC de abdomen y pelvis mostró voluminosa formación heterogénea de aspecto sólido sin plano de clivaje con respecto al parénquima hepático e intenso realce con contraste. Luego de la extirpación de la masa retroperitoneal, evolucionó con valores de glucemia y potasemia normales, estabilizó la presión arterial y recuperó los ciclos menstruales.

  13. La curiosidad por la naturaleza

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    José Vicente Rodríguez

    2016-07-01

    Full Text Available Esta actitud es un instrumento estratégico para estimular procesos de desarrollo realmente sostenibles, donde se aproveche la admiración que nos embarga al conocer y apreciar lo que nos rodea para responsablemente utilizar y conservar nuestros recursos naturales. Con esta reflexión deseo resaltar esa motivación de escudriñar lo desconocido, que fue también la virtud que propició el desarrollo de la personalidad de Francisco José de Caldas y Tenorio, uno de nuestros primeros y connotados sabios colombianos, y en cuyo honor se constituyó la revista de divulgación científica más importante del país, que en estos momentos cumple 76 años de existencia. Caldas tuvo curiosidad por todo lo que existía en su entorno; inicialmente en su natal Popayán, donde se maravilló por lo natural, lo que lo motivó y llevó más allá de su tierra a formar parte de la mayor empresa de exploración científica adelantada en ese entonces en territorio americano: la primera Expedición Botánica al nuevo Reino de Granada, de José Celestino Mutis.

  14. Mastite bovina por Mycoplasma bovis em rebanhos leiteiros Mastitis caused by Mycoplasma bovis in dairy cattle

    Directory of Open Access Journals (Sweden)

    Lucienne G. Pretto

    2001-12-01

    Full Text Available Foram examinadas 713 vacas de três rebanhos leiteiros localizados na região norte do Estado do Paraná e sudoeste do Estado de São Paulo, das quais 137 apresentaram mastite. Nas três propriedades foram detectados oito animais (1,12% com mastite clínica por Mycoplasma bovis. Destes animais, quatro tratados com oxitetraciclina e tilosina e três com enrofloxacina, não responderam ao tratamento e foram descartados no decorrer da lactação. Uma vaca medicada com enrofloxacina recuperou quase que totalmente a secreção láctea mas a eliminação de M. bovis persistiu por toda lactação. Esta vaca apresentou cura bacteriológica na lactação seguinte. O descarte dos animais positivos, monitora-mento bacteriológico e a aplicação correta das medidas de prevenção para as mastites contagiosas controlaram a disseminação de M. bovis nos rebanhos.In this study 713 cows were examined. The animals were from three dairy farms in northern Paraná and the southwest of the State of São Paulo. From these cows, 137 had mastitis. On the three farms, 8 cows (1.12% with Mycoplasma bovis mastitis were detected. Four were treated with tylosin and oxytetracyclin and three with enrofloxacin. There was no response to the treatments, and these animals were culled during the lactation period. One cow treated with enrofloxacin almost totally recovered milk production, but elimination of M. bovis continued during the lactation, and there was no bacteriological cure. This cow had a normal milk production in the next lactation period, without elimination of M. bovis. Culling of positive animals, the bacteriological study and correct application of preventive practices for contagious mastitis controlled the dissemination of M. bovis to other animals.

  15. Mortalidad por meningitis por Pasteurella canis. Oportunidades de aprendizaje

    Directory of Open Access Journals (Sweden)

    Ana Rosa Ropero Vera

    2016-01-01

    Full Text Available La meningitis bacteriana es una enfermedad importante de distribución mundial, causa mayor y sustancial de mortalidad y morbilidad en países en desarrollo. La Organización Mundial de la Salud (OMS sostiene que la meningitis es una de las diez afecciones principales del ser humano y debe ser considerada como una emergencia infectológica; por eso es fundamental reconocer que esta enfermedad es causa de muerte en niños de todo el mundo, sin distinción de raza, nivel económico o sociocultural. Se realizó una investigación de caso en menor de 53 días de nacido, que cumplía con los criterios clínicos y de laboratorio compatible con meningitis bacteriana, con el propósito de analizar y fortalecer la toma de decisiones en salud pública por parte de la secretaría local de salud del municipio de Valledupar (Colombia. Entre los hallazgos se encontró antecedentes infecciosos en el menor, coloración de Gram y cultivo de LCR, en el que se identificó cocobacilos Gram negativos, que fueron aislados como agente causal Pasteurella canis. Este estudio pretende sensibilizar a los prestadores de salud para que cuenten con personal altamente capacitado para brindar tratamientos adecuados y prevenir complicaciones en la meningitis bacteriana en niños, y así disminuir la posibilidad de secuelas o muerte, tanto en pacientes con compromiso inmunológico o sin este.

  16. Mortalidad intrahospitalaria por accidente cerebrovascular

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    Federico Rodríguez Lucci

    2013-08-01

    Full Text Available La mortalidad global por accidente cerebrovascular (ACV ha disminuido en las últimas tres décadas, probablemente debido a un mejor control de los factores de riesgo vascular. La mortalidad hospitalaria por ACV ha sido tradicionalmente estimada entre 6 y 14% en la mayoría de las series comunicadas. Sin embargo, los datos de ensayos clínicos recientes sugieren que esta cifra sería sustancialmente menor. Se revisaron datos de pacientes internados con diagnóstico de ACV del Banco de Datos de Stroke de FLENI y los registros institucionales de mortalidad entre los años 2000 y 2010. Los subtipos de ACV isquémicos se clasificaron según criterios TOAST y los ACV hemorrágicos en hematomas intrapanquimatosos, hemorragias subaracnoideas aneurismáticas, malformaciones arteriovenosas y otros hematomas intraparenquimatosos. Se analizaron 1514 pacientes, 1079 (71% con ACV isquémico (grandes vasos 39%, cardioembólicos 27%, lacunares 9%, etiología indeterminada 14%, otras etiologías 11% y 435 (29% con ACV hemorrágico (intraparenquimatosos 27%, hemorragia subaracnoidea 30%, malformaciones arteriovenosas 25% y otros hematomas espontáneos 18%. Se registraron 38 muertes intrahospitalarias (17 ACV isquémicos y 21 ACV hemorrágicos, representando una mortalidad global del 2.5% (1.7% en ACV isquémicos y 4.8% en ACV hemorrágicos. No se registraron muertes asociadas al uso de fibrinolíticos endovenosos. La mortalidad intrahospitalaria en pacientes con ACV isquémico y hemorrágico en nuestro centro fue baja. El manejo en un centro dedicado a las enfermedades neurológicas y el enfoque multidisciplinario por personal médico y no médico entrenado en el cuidado de la enfermedad cerebrovascular podrían explicar, al menos en parte, estos resultados.

  17. Spatial patterns of tree community dynamics are detectable in a small (4 ha and disturbed fragment of the Brazilian Atlantic forest Padrões espaciais de dinâmicas da comunidade de arbórea num fragmento pequeno (4 ha e perturbado podem ser detectados

    Directory of Open Access Journals (Sweden)

    Evandro Luiz Mendonça Machado

    2010-03-01

    ambiental de um fragmento florestal de área pequena (4,0 ha. Foram obtidas taxas de mortalidade e recrutamento de árvores e taxas de ganho e perda de área basal para a amostra total, quatro habitats de solo previamente definidos, classes de diâmetro e populações. A comunidade arbórea mostrou-se instável no período, uma vez que as taxas de mortalidade superaram as de recrutamento e as taxas de perda superaram as de ganho em área basal tanto na amostra total como nos habitats de solo. Tais mudanças gerais se relacionaram, possivelmente, a uma fase de degradação do ciclo silvigenético provavelmente desencadeada por um severo episódio de distúrbio ocorrido no passado. A dinâmica da comunidade não foi homogênea em todo o fragmento, diferiu significativamente entre os habitats de solo. As variáveis ambientais que se correlacionaram mais fortemente com a variação das taxas de dinâmica foram aquelas vinculadas à disponibilidade de água, luz e nutrientes minerais. Em contraste com a tendência global, as espécies de subdossel expandiram em densidade no período, provavelmente em resposta ao mesmo evento de distúrbio.

  18. Genotipificación de Plasmodium falciparum por PCR múltiple por medio de los genes msp1, msp2 y glurp, en cuatro localidades de Colombia

    Directory of Open Access Journals (Sweden)

    Sandra Milena Barrera

    2010-12-01

    Conclusiones. Nuestros resultados demuestran que el marcador molecular msp1 no proporciona información útil para diferenciar las poblaciones parasitarias de P. falciparum. El gen msp2 es apropiado para evaluar la diversidad genética, pero requiere ensayos más finos que permitan diferenciar claramente el polimorfismo de tamaño en las dos familias. Los resultados obtenidos con el gen glurp evidenciaron una gran diversidad genética en las poblaciones de P. falciparum que circulan en el país y sugieren que este gen puede ser útil para diferenciar nuevas infecciones de infecciones recurrentes o recrudecimientos.

  19. Species identification and quantification in meat and meat products using droplet digital PCR (ddPCR).

    Science.gov (United States)

    Floren, C; Wiedemann, I; Brenig, B; Schütz, E; Beck, J

    2015-04-15

    Species fraud and product mislabelling in processed food, albeit not being a direct health issue, often results in consumer distrust. Therefore methods for quantification of undeclared species are needed. Targeting mitochondrial DNA, e.g. CYTB gene, for species quantification is unsuitable, due to a fivefold inter-tissue variation in mtDNA content per cell resulting in either an under- (-70%) or overestimation (+160%) of species DNA contents. Here, we describe a reliable two-step droplet digital PCR (ddPCR) assay targeting the nuclear F2 gene for precise quantification of cattle, horse, and pig in processed meat products. The ddPCR assay is advantageous over qPCR showing a limit of quantification (LOQ) and detection (LOD) in different meat products of 0.01% and 0.001%, respectively. The specificity was verified in 14 different species. Hence, determining F2 in food by ddPCR can be recommended for quality assurance and control in production systems. Copyright © 2014 The Authors. Published by Elsevier Ltd.. All rights reserved.

  20. Herida precordial por arma blanca

    OpenAIRE

    Marcelo F. Jiménez; Nuria Novoa; José Luis Aranda; Gonzalo Varela

    2005-01-01

    Varón de 42 años con esquizofrenia paranoide. Intento de suicidio con tres autolesiones por arma blanca (Fig. 1). Fue traído al hospital en estado consciente, taquicárdico, taquipneico y con tensión arterial (TA) de 140/90 mmHg. Fue trasladado al quirófano, donde se practicó una toracotomía axilar izquierda sin extraer el arma. Durante la intervención se apreciaron tres heridas incisas, dos no penetrantes, hemotórax de 150 ml, herida pericárdica de 4 cm, sin lesiones cardíacas. La hoja del ar...

  1. Human Papillomavirus infection in men residing in Brazil, Mexico, and the USA Infección por Virus de Papiloma Humano en hombres de Brasil, México y EUA

    Directory of Open Access Journals (Sweden)

    2008-10-01

    Virus del Papiloma Humano (VPH entre hombres de 18 años o más de tres países con un protocolo común para el muestreo de la detección de VPH, y evaluar si la detección de VPH varía de acuerdo con la edad y el país. MATERIAL Y MÉTODOS: El estudio incluye diversas etapas que inician con la identificación de hombres susceptibles, una medición basal (visita de enrolamiento y nueve visitas adicionales programadas cada seis meses. En este artículo, se presenta el análisis de los primeros 1160 hombres que fueron incluídos en el estudio. Para maximizar la posibilidad de detección de VPH se utilizó un cepillo de dacrón que muestreó en forma combinada diferentes sitios anatómicos. Para la determinación de ADN de VPH se utilizó ión en cadena de polimerasa (PCR por amplificación de un fragmento del gen de VPH L1. RESULTADOS: Entre 1160 hombres de Brasil, México y EUA, la prevalencia global de VPH fue de 65.2%, con solamente 12% de tipos oncogénicos, 20.7% de tipos de VPH no oncogénicos, 17.8% de muestras positivas a tipos oncogénicos y no oncogénicos; y finalmente 14.7% de infecciones no clasificadas. Múltiples tipos de VPH fueron detectados en 25.7% de los participantes en el estudio. La prevalencia de VPH fue más alta en Brasil (72.3%, comparada con la observada en EUA (61.3% y México (61.9%. Los tipos de VPH 16 (6.5%, 51 (6.5% y 59 (5.3% fueron los más comúnmente observados con poder oncogénico. El VPH 84 (7.7%, 62 (7.3% y 6 (6.6% fueron las infecciones no oncogénicas más comunes. CONCLUSIONES: Son necesarios estudios de la distribución de VPH en un amplio margen de edad entre hombres de múltiples países, para establecer con mayor precisión, el conocimiento de la historia natural de la infección por VPH en hombres.

  2. High quality DNA from human papillomavirus (HPV for PCR/RFLPs

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    Denise Wanderlei-Silva

    2005-01-01

    Full Text Available The analysis of DNA in clinical samples for a secure diagnostic has become indispensable nowadays. Techniques approaching isolation of high molecular weigth DNA of HPV could lead to efficient amplification and early clinical diagnosis of the virus DNA by PCR (polymerase chain reaction. We describe a fast, non-toxical, efficient and cheap method for DNA isolation of human papilloma virus (HPV from cervical smears using guanidine (DNAzol solution. A 450 bp DNA band correponding to the late region (L1 of the virus genome was detected by PCR, showing that the DNAzol extraction soluction generated a good viral DNA yield. The electrophoretic pattern after digestion with restriction endonucleases (RFLPs/PCR revealed the predominance of HPV-16 and HPV-33 in the samples from the State of Alagoas, Brazil.A detecção de DNA em amostras clínicas visando um diagnóstico mais seguro vem se tornando uma prática comum em laboratórios de análise clínica. Metodologias que objetivem o isolamento de DNA de alto peso molecular de HPV podem levar a uma amplificação precisa e diagnose precoce do DNA do vírus por PCR (reação de polimerase em cadeia. Nós descrevemos um método para o isolamento do DNA do vírus do papiloma humano de amostras cervicais utilizando o detergente guanidina (solução DNAzol. O método foi rápido, não-tóxico e eficiente. Uma banda de DNA de 450 pb correspondente à região tardia (L1 do genoma viral foi detectada por PCR, mostrando que a extração com DNAzol gerou quantidade suficiente de DNA para análise. O padrão eletroforético, após digestão com endonucleases de restrição (RFLPs/PCR, revelou predominância de HPV 16 e HPV-33 nas amostras no Estado de Alagoas, Brasil.

  3. Analysis of extracellular RNA by digital PCR

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    Kenji eTakahashi

    2014-06-01

    Full Text Available The transfer of extracellular RNA is emerging as an important mechanism for intracellular communication. The ability for the transfer of functionally active RNA molecules from one cell to another within vesicles such as exosomes enables a cell to modulate cellular signaling and biological processes within recipient cells. The study of extracellular RNA requires sensitive methods for the detection of these molecules. In this methods article, we will describe protocols for the detection of such extracellular RNA using sensitive detection technologies such as digital PCR. These protocols should be valuable to researchers interested in the role and contribution of extracellular RNA to tumor cell biology.

  4. Determining Fungi rRNA Copy Number by PCR

    Science.gov (United States)

    The goal of this project is to improve the quantification of indoor fungal pollutants via the specific application of quantitative PCR (qPCR). Improvement will be made in the controls used in current qPCR applications. This work focuses on the use of two separate controls within ...

  5. Real-Time PCR for Universal Phytoplasma Detection and Quantification

    DEFF Research Database (Denmark)

    Christensen, Nynne Meyn; Nyskjold, Henriette; Nicolaisen, Mogens

    2013-01-01

    Currently, the most efficient detection and precise quantification of phytoplasmas is by real-time PCR. Compared to nested PCR, this method is less sensitive to contamination and is less work intensive. Therefore, a universal real-time PCR method will be valuable in screening programs and in other...

  6. Comparison of a Commercially Available Repetitive-Element PCR System (DiversiLab) with PCR Ribotyping for Typing of Clostridium difficile Strains ▿

    OpenAIRE

    Eckert, C.; Van Broeck, J.; Spigaglia, P.; Burghoffer, B.; Delmée, M.; Mastrantonio, P.; Barbut, F.

    2011-01-01

    This study compared a repetitive-element PCR (rep-PCR) method (DiversiLab system) to PCR ribotyping. The discriminatory power of rep-PCR was 0.997. Among the PCR ribotype 027 isolates tested, different rep types could be distinguished. rep-PCR showed a higher discriminatory power than PCR ribotyping. Nevertheless, this method requires technical skill, and visual interpretation of rep-PCR fingerprint patterns may be difficult.

  7. Automated PCR setup for forensic casework samples using the Normalization Wizard and PCR Setup robotic methods.

    Science.gov (United States)

    Greenspoon, S A; Sykes, K L V; Ban, J D; Pollard, A; Baisden, M; Farr, M; Graham, N; Collins, B L; Green, M M; Christenson, C C

    2006-12-20

    Human genome, pharmaceutical and research laboratories have long enjoyed the application of robotics to performing repetitive laboratory tasks. However, the utilization of robotics in forensic laboratories for processing casework samples is relatively new and poses particular challenges. Since the quantity and quality (a mixture versus a single source sample, the level of degradation, the presence of PCR inhibitors) of the DNA contained within a casework sample is unknown, particular attention must be paid to procedural susceptibility to contamination, as well as DNA yield, especially as it pertains to samples with little biological material. The Virginia Department of Forensic Science (VDFS) has successfully automated forensic casework DNA extraction utilizing the DNA IQ(trade mark) System in conjunction with the Biomek 2000 Automation Workstation. Human DNA quantitation is also performed in a near complete automated fashion utilizing the AluQuant Human DNA Quantitation System and the Biomek 2000 Automation Workstation. Recently, the PCR setup for casework samples has been automated, employing the Biomek 2000 Automation Workstation and Normalization Wizard, Genetic Identity version, which utilizes the quantitation data, imported into the software, to create a customized automated method for DNA dilution, unique to that plate of DNA samples. The PCR Setup software method, used in conjunction with the Normalization Wizard method and written for the Biomek 2000, functions to mix the diluted DNA samples, transfer the PCR master mix, and transfer the diluted DNA samples to PCR amplification tubes. Once the process is complete, the DNA extracts, still on the deck of the robot in PCR amplification strip tubes, are transferred to pre-labeled 1.5 mL tubes for long-term storage using an automated method. The automation of these steps in the process of forensic DNA casework analysis has been accomplished by performing extensive optimization, validation and testing of the

  8. Reação em cadeia da polimerase para detecção de Clostridium chauvoei em tecidos de Cavia porcellus PCR detection of Clostridium chauvoei in tissues of Cavia porcellus

    Directory of Open Access Journals (Sweden)

    Ronnie Antunes de Assis

    2005-12-01

    Full Text Available O objetivo deste trabalho foi padronizar uma técnica de reação em cadeia da polimerase (PCR, para detecção de Clostridium chauvoei, em tecidos fixados em formol e incluídos em parafina de cobaias (Cavia porcellus experimentalmente infectadas com esse microrganismo. Os animais foram sacrificados, e amostras do músculo da área de inoculação (MAI, fígado, miocárdio e baço foram disponibilizadas para a técnica de PCR. O clostrídio foi detectado em todas as secções do MAI, fígado e miocárdio, mas não foi observado em secções do baço. Reações cruzadas não foram observadas a partir de secções do MAI dos animais com inoculação de outras espécies de clostrídios, bem como nenhuma amplificação foi observada a partir de secções do MAI dos animais controle. Esses resultados mostram que a técnica de PCR desenvolvida neste estudo, pode ser usada para detecção de Clostridium chauvoei em tecidos fixados em formol e incluídos em parafina.The objective of this work was the standardization of a polymerase chain reaction (PCR for detection of Clostridium chauvoei in formalin-fixed, paraffin-embedded tissues of guinea-pigs (Cavia porcellus infected experimentally with this microorganism. The animals were sacrificed, and samples of muscle from inoculation area (MIA, liver, myocardium and spleen were available for PCR technique. Clostridium chauvoei was detected in all sections of the MIA, liver and myocardium, and no product was observed in sections of the spleen. Cross-reactions were not observed in sections of MIA of the animals inoculated with other clostridia, as well as no amplification was observed in sections of MIA of control animals. These results show that the PCR technique developed in this study may be useful for detection of C. chauvoei in formalin-fixed, paraffin-embedded tissues.

  9. Comparison of nested PCR and qPCR for the detection and quantitation of BoHV6 DNA.

    Science.gov (United States)

    Kubiś, Piotr; Materniak, Magdalena; Kuźmak, Jacek

    2013-12-01

    Nested PCR and qPCR (quantitative PCR) tests based on glycoprotein B (gB) gene were designed for detecting Bovine herpesvirus 6 (BoHV6) in bovine whole blood samples and wild ruminant blood clots (deer and roe-deer). This virus, commonly known as BLHV (bovine lymphotropic herpesvirus) belongs to the Herpesviridae family, subfamily Gammaherpesvirinae and Macavirus genus. DNA isolated from 92 dairy cow blood samples and 69 wild ruminant clots were examined for the presence of BoHV6 using nested PCR and qPCR tests. Viral DNA was detected by using nested PCR in 59 out of 92 bovine blood samples (64.1%), and by qPCR in 68 out of 92 bovine blood samples (73.9%), but none out of 69 DNA samples isolated from wild ruminant blood clots, was positive in both assays. The specificity of nested PCR and qPCR was confirmed by using BoHV1, BoHV4, BoHV6, BFV, BIV, and BLV DNA. The sensitivity of nested PCR and qPCR was determined using a serially 10-fold diluted vector pCR2.1HgB (2 × 10(0)-2 × 10(6)copies/reaction). In this testing, qPCR was more sensitive than the nested PCR, detecting two copies of BoHV6 whilst the limit of detection for nested PCR was 20 copies. In all qPCR assays, the coefficients of determination (R(2)) ranged between 0.990 and 0.999, and the calculated amplification efficiencies (Eff%) within the range of 89.7-106.9. The intra- and inter-assay CV (coefficient of variation) values did not exceed 4%. Copyright © 2013 Elsevier B.V. All rights reserved.

  10. Identification of periodontopathogen microorganisms by PCR technique

    Directory of Open Access Journals (Sweden)

    Milićević Radovan

    2008-01-01

    Full Text Available INTRODUCTION Periodontitis is an inflammatory disease of the supporting tissues of teeth and is a major cause of tooth loss in adults. The onset and progression of periodontal disease is attributed to the presence of elevated levels of a consortium of pathogenic bacteria. Gram negative bacteria, mainly strict anaerobes, play the major role. OBJECTIVE The present study aimed to assess the presence of the main types of microorganisms involved in the aetiopathogenesis of periodontal disease: Porphyromonas gingivalis, Aggregatibacter actinomycetemcomitans, Eikenella corrodens, Treponema denticola, Tanerella forsythia and Prevotella intermedia in different samples collected from the oral cavity of 90 patients diagnosed with periodontitis. METHOD Bacterial DNA detection was performed in diverse biological materials, namely in dental plaque, gingival tissue and saliva, by means of multiplex PCR, a technique that allows simultaneous identification of two different bacterial genomes. RESULTS In the dental plaque of the periodontitis patients, Treponema denticola dominated. In the gingival tissue, Tannerella forsythia and Treponema denticola were the microbiota most frequently detected, whilst in saliva Treponema denticola and Eikenella corrodens were found with the highest percentage. CONCLUSION The identification of microorganisms by multiplex PCR is specific and sensitive. Rapid and precise assessment of different types of periodontopathogens is extremely important for early detection of the infection and consequently for the prevention and treatment of periodontal disease. In everyday clinical practice, for routine bacterial evaluation in patients with periodontal disease, the dental plaque is the most suitable biological material, because it is the richest in periodontal bacteria.

  11. Denoising PCR-amplified metagenome data

    Directory of Open Access Journals (Sweden)

    Rosen Michael J

    2012-10-01

    Full Text Available Abstract Background PCR amplification and high-throughput sequencing theoretically enable the characterization of the finest-scale diversity in natural microbial and viral populations, but each of these methods introduces random errors that are difficult to distinguish from genuine biological diversity. Several approaches have been proposed to denoise these data but lack either speed or accuracy. Results We introduce a new denoising algorithm that we call DADA (Divisive Amplicon Denoising Algorithm. Without training data, DADA infers both the sample genotypes and error parameters that produced a metagenome data set. We demonstrate performance on control data sequenced on Roche’s 454 platform, and compare the results to the most accurate denoising software currently available, AmpliconNoise. Conclusions DADA is more accurate and over an order of magnitude faster than AmpliconNoise. It eliminates the need for training data to establish error parameters, fully utilizes sequence-abundance information, and enables inclusion of context-dependent PCR error rates. It should be readily extensible to other sequencing platforms such as Illumina.

  12. Miotoxicidade por organofosforados Organophosphate myotoxicity

    Directory of Open Access Journals (Sweden)

    Maria J. Cavaliere

    1996-06-01

    Full Text Available Os organofosforados são um grupo de compostos químicos amplamente utilizados em agropecuária como inseticidas, ocasionando intoxicações acidentais em animais e humanos, e mesmo sendo utilizados em tentativas de suicídio. A toxicidade desses produtos decorre sobretudo de insuficiência cárdio-respiratória por compromentimento do sistema nervoso autônomo. Sabe-se que alguns destes compostos induzem em animais de experimentação e em humanos, uma miopatia caracterizada por degeneração de células musculares, comprometendo sobretudo a musculatura respiratória. Baseado no fato de que este comprometimento contribui para a piora da função respiratória, propõe-se um protocolo de avaliação rotineira de miotoxicidade por compostos organofosforados, através de uma bateria mínima e suficiente de colorações e reações histoquímicas para quantificação da necrose muscular. Utilizaram-se como modelo experimental, grupos de ratos albinos (Wistar intoxicados com o organofosforado paraoxon, com e sem antídotos (atropina ou pralidoxima. Verificou-se nos grupos tratados com paraoxon e paraoxon mais atropina, necrose de fibras musculares no diafragma, que atingia em determinadas áreas até 15% das fibras. No grupo tratado com paraoxon mais pralidoxima, a necrose foi mínima, evidenciando o papel mioprotetor deste último antídoto.Organophosphates comprise a group of chemical compounds extensively used in farming as insecticides, which cause accidental poisoning in animals and men and are also used in suicide attempts. The toxicity of these compounds is due especially to the cardiac and respiratory impairment in consequence of autonomic nervous system disorders. However, it is known that some of these products induce a myopathy in experimental animals and humans. This myopathy is characterized by muscle cell degeneration, involving above all the respiratory muscles. Based on the fact that this involvement certainly enhances the

  13. Primer reporte de un caso importado de Malaria por Plasmodium ovale curtisi en Paraguay, confirmado por diagnóstico molecular

    Directory of Open Access Journals (Sweden)

    Florencia del Puerto

    2015-04-01

    Full Text Available En países donde el Plasmodium ovale no es común, los microscopistas tienden a identificarlo de manera errónea como Plasmodium vivax. En este trabajo reportamos la identificación de la especie P. ovale curtisi por el método de PCR múltiple semianidada (SnM-PCR y la secuenciación de la subunidad pequeña del gen del ARN 18S, en un paciente paraguayo de 44 años de edad que vino en el 2.013 de Guinea Ecuatorial, África Occidental, a quien se le diagnosticó una infección por P. vivax por microscopía convencional. El empleo de métodos moleculares para la identificación de casos importados de infección con especies del género Plasmodium es uno de los objetivos principales en el control y la prevención de la malaria en Paraguay, teniendo en cuenta que el país se encuentra en fase de pre-eliminación de la enfermedad.

  14. PCR en tiempo real para el sexaje y análisis citológico de la sinapsis del bivalente más pequeño en espermatocitos en paquiteno de Oreochromis niloticus

    Directory of Open Access Journals (Sweden)

    Zulita Prieto

    2017-01-01

    Full Text Available El objetivo de la presente investigación fue dar a conocer nuevos marcadores moleculares para la PCR en tiempo real asociados a los cromosomas sexuales y el análisis de la sinapsis del bivalente más pequeño en espermatocitos en paquiteno de Oreochromis nil oticus . Se diseñaron cuatro pares de cebadores y se pusieron a prueba por PCR punto final y por PCR en tiempo real utilizando el fluorescente SYBR® Green en muestras de ADN de hembras XX, machos XY y supermachos YY. Los tamaños de los fragmentos amplificad os por PCR punto final fueron concordantes a los valores esperados y por PCR en tiempo real se demostró igual especificidad pero con ventaja en rapidez en la detección de amplificación de marcadores asociados a los cromosoma X y Y, de acuerdo con las condi ciones de la PCR establecidas. Las diferencias genéticas entre las regiones de los cromosomas X y Y demostradas con los marcadores específicos no fueron perceptibles citológicamente en los espermatocitos en paquiteno de O. niloticus XY. En base a las difer encias y homologías de las secuencias de ADN entre las accesiones KC710223 y KC710224 de los cromosomas X y Y se propone un modelo de sinapsis del bivalente XY.

  15. Efecto del acibenzolar-s-metil sobre el desarrollo de la virosis causada por potyvirus en tomate de árbol

    Directory of Open Access Journals (Sweden)

    Mejía A. Diana Marcela

    2009-04-01

    Full Text Available

    El principal limitante del cultivo de tomate de árbol en Colombia es el ataque de virus, el cual reduce la producción y calidad de la fruta. En condiciones de invernadero se evaluó el efecto del inductor de resistencia Acibenzolar-S-metil- ASM (Boost® sobre la incidencia y severidad de la virosis en plantas inoculadas con extracto de hojas naturalmente infectadas con virus, mediante aplicación foliar de 0,08% de producto comercial por planta cada 20 días. La incidencia de la virosis se redujo en 50% cuando el inductor se aplicó antes de la inoculación con el virus, seguido de dos aplicaciones adicionales de ASM, comparado con 100% de incidencia en las plantas inoculadas solo con el extracto de plantas infectadas con el virus, tendencia encontrada también en la curva de desarrollo de la enfermedad. Se observó un retraso de 7 días en la aparición de síntomas cuando se aplicó el inductor antes de la inoculación así como se observó una reducción significativa en el grado de severidad de la enfermedad. El potyvirus fue detectado mediante la prueba ELISA; sin embargo, no se encontraron diferencias estadísticas significativas en su concentración entre los diferentes tratamientos.

  16. Reparación de hernias inguinales recidivantes por vía preperitoneal con el uso de mallas protésicas

    Directory of Open Access Journals (Sweden)

    Manuel Espinel González

    1997-04-01

    Full Text Available Se analizaron los resultados del tratamiento quirúrgico de 17 pacientes, afectados de hernias inguinales con 2 o más recidivas, en 3 de ellos las hernias eran bilaterales. Se utilizó para su reparación mallas sintéticas de polyester (mersilene y de polipropilene (marlex, las que fueron colocadas por vía abdominal preperitoneal. Después de un período de seguimiento de 1 a 4 años no se ha detectado recidiva herniaria en ningún pacienteResult of the surgical treatment on 17 patients presenting with inguinal hernia with two or more recurrences were analyzed. In 3 of them the hernia was bilateral. Synthetic polyester surgical mesh (mersilene and a polypropilene mesh (marlex were used for the repair. The surgical mesh was placed by preperitoneal abdominal via. After a follow-up period of 1-4 years no recurrence has been found in none of the patients

  17. Herida precordial por arma blanca

    Directory of Open Access Journals (Sweden)

    Marcelo F. Jiménez

    2005-04-01

    Full Text Available Varón de 42 años con esquizofrenia paranoide. Intento de suicidio con tres autolesiones por arma blanca (Fig. 1. Fue traído al hospital en estado consciente, taquicárdico, taquipneico y con tensión arterial (TA de 140/90 mmHg. Fue trasladado al quirófano, donde se practicó una toracotomía axilar izquierda sin extraer el arma. Durante la intervención se apreciaron tres heridas incisas, dos no penetrantes, hemotórax de 150 ml, herida pericárdica de 4 cm, sin lesiones cardíacas. La hoja del arma blanca penetraba a través del diafragma produciendo una herida incisa en el lóbulo hepático izquierdo (Fig. 2. Se extrajo el arma y se suturó la laceración hepática. El paciente fue trasladado el cuarto día a la Unidad de Psiquiatría.

  18. por láser

    Directory of Open Access Journals (Sweden)

    Mayra Garcimuño

    2013-01-01

    Full Text Available En el presente trabajo, la técnica Espectroscopia de plasmas producidos por láser (Laser-induced breakdown spectroscopy –LIBS– se aplicó a la determinación cuan- titativa de Na en agua natural dulce, de interés en agricultura para el estudio de la alcalinidad de aguas de regadío. Para efectuar el análisis, se prepararon soluciones con concentraciones conocidas del analito, se mezclaron con óxido de calcio y se compactaron en pastillas sólidas. Los plasmas se produjeron en aire a presión atmos- férica utilizando un láser pulsado Nd:YAG. Se construyó una curva de calibración y se calculó el límite de detección. Se analizaron muestras de agua natural y los resultados se compararon con los obtenidos mediante espectroscopia de absorción atómica. Se demostró la factibilidad del método para la determinación de Na en agua natural dulce.

  19. Accurate quantification of supercoiled DNA by digital PCR

    Science.gov (United States)

    Dong, Lianhua; Yoo, Hee-Bong; Wang, Jing; Park, Sang-Ryoul

    2016-01-01

    Digital PCR (dPCR) as an enumeration-based quantification method is capable of quantifying the DNA copy number without the help of standards. However, it can generate false results when the PCR conditions are not optimized. A recent international comparison (CCQM P154) showed that most laboratories significantly underestimated the concentration of supercoiled plasmid DNA by dPCR. Mostly, supercoiled DNAs are linearized before dPCR to avoid such underestimations. The present study was conducted to overcome this problem. In the bilateral comparison, the National Institute of Metrology, China (NIM) optimized and applied dPCR for supercoiled DNA determination, whereas Korea Research Institute of Standards and Science (KRISS) prepared the unknown samples and quantified them by flow cytometry. In this study, several factors like selection of the PCR master mix, the fluorescent label, and the position of the primers were evaluated for quantifying supercoiled DNA by dPCR. This work confirmed that a 16S PCR master mix avoided poor amplification of the supercoiled DNA, whereas HEX labels on dPCR probe resulted in robust amplification curves. Optimizing the dPCR assay based on these two observations resulted in accurate quantification of supercoiled DNA without preanalytical linearization. This result was validated in close agreement (101~113%) with the result from flow cytometry. PMID:27063649

  20. Osteomalacia inducida por tumor: hemangiopericitoma rinosinusal

    Directory of Open Access Journals (Sweden)

    Enriqueta M. Serafini

    2013-02-01

    Full Text Available La osteomalacia inducida por tumor es una rara enfermedad del metabolismo óseo caracterizada por el aumento en la excreción de fosfato a nivel renal seguido de hipofosfatemia. Es causada por agentes fosfatúricos producidos por determinados tumores. La resección total del tumor resulta en la completa reversión de las anormalidades bioquímicas, la desaparición de las manifestaciones clínicas y los hallazgos en los estudios por imágenes. Presentamos el caso de un varón de 61 años con cuadro clínico y laboratorio compatibles con osteomalacia oncogénica inducida por tumor mesenquimático de localización rinosinusal. En nuestro caso el diagnóstico histológico correspondió a una neoplasia de tipo vascular: hemangiopericitoma.

  1. A survey of tools for the analysis of quantitative PCR (qPCR data

    Directory of Open Access Journals (Sweden)

    Stephan Pabinger

    2014-09-01

    Our comprehensive survey showed that most tools use their own file format and only a fraction of the currently existing tools support the standardized data exchange format RDML. To allow a more streamlined and comparable analysis of qPCR data, more vendors and tools need to adapt the standardized format to encourage the exchange of data between instrument software, analysis tools, and researchers.

  2. Simultaneous Detection of Ricin and Abrin DNA by Real-Time PCR (qPCR

    Directory of Open Access Journals (Sweden)

    Roman Wölfel

    2012-08-01

    Full Text Available Ricin and abrin are two of the most potent plant toxins known and may be easily obtained in high yield from the seeds using rather simple technology. As a result, both toxins are potent and available toxins for criminal or terrorist acts. However, as the production of highly purified ricin or abrin requires sophisticated equipment and knowledge, it may be more likely that crude extracts would be used by non-governmental perpetrators. Remaining plant-specific nucleic acids in these extracts allow the application of a real-time PCR (qPCR assay for the detection and identification of abrin or ricin genomic material. Therefore, we have developed a duplex real-time PCR assays for simultaneous detection of ricin and abrin DNA based on the OmniMix HS bead PCR reagent mixture. Novel primers and hybridization probes were designed for detection on a SmartCycler instrument by using 5′-nuclease technology. The assay was thoroughly optimized and validated in terms of analytical sensitivity. Evaluation of the assay sensitivity by probit analysis demonstrated a 95% probability of detection at 3 genomes per reaction for ricin DNA and 1.2 genomes per reaction for abrin DNA. The suitability of the assays was exemplified by detection of ricin and abrin contaminations in a food matrix.

  3. Analytical Performance of Four Polymerase Chain Reaction (PCR and Real Time PCR (qPCR Assays for the Detection of Six Leishmania Species DNA in Colombia

    Directory of Open Access Journals (Sweden)

    Cielo M. León

    2017-10-01

    Full Text Available Leishmaniasis comprises a spectrum of parasitic diseases caused by protozoans of the genus Leishmania. Molecular tools have been widely employed for the detection of Leishmania due to its high sensitivity and specificity. However, the analytical performance of molecular platforms as PCR and real time PCR (qPCR including a wide variety of molecular markers has never been evaluated. Herein, the aim was to evaluate the analytical performance of 4 PCR-based assays (designed on four different targets and applied on conventional and real-time PCR platforms. We evaluated the analytical performance of conventional PCR and real time PCR, determining exclusivity and inclusivity, Anticipated Reportable Range (ARR, limit of detection (LoD and accuracy using primers directed to kDNA, HSP70, 18S and ITS-1 targets. We observed that the kDNA was the most sensitive but does not meet the criterion of exclusivity. The HSP70 presented a higher LoD in conventional PCR and qPCR in comparison with the other markers (1 × 101 and 1 × 10-1 equivalent parasites/mL respectively and had a higher coefficient of variation in qPCR. No statistically significant differences were found between the days of the test with the four molecular markers. The present study revealed that the 18S marker presented the best performance in terms of analytical sensitivity and specificity for the qPCR in the species tested (species circulating in Colombia. Therefore, we recommend to explore the analytical and diagnostic performance in future studies using a broader number of species across America.

  4. Analytical Performance of Four Polymerase Chain Reaction (PCR) and Real Time PCR (qPCR) Assays for the Detection of Six Leishmania Species DNA in Colombia

    Science.gov (United States)

    León, Cielo M.; Muñoz, Marina; Hernández, Carolina; Ayala, Martha S.; Flórez, Carolina; Teherán, Aníbal; Cubides, Juan R.; Ramírez, Juan D.

    2017-01-01

    Leishmaniasis comprises a spectrum of parasitic diseases caused by protozoans of the genus Leishmania. Molecular tools have been widely employed for the detection of Leishmania due to its high sensitivity and specificity. However, the analytical performance of molecular platforms as PCR and real time PCR (qPCR) including a wide variety of molecular markers has never been evaluated. Herein, the aim was to evaluate the analytical performance of 4 PCR-based assays (designed on four different targets) and applied on conventional and real-time PCR platforms. We evaluated the analytical performance of conventional PCR and real time PCR, determining exclusivity and inclusivity, Anticipated Reportable Range (ARR), limit of detection (LoD) and accuracy using primers directed to kDNA, HSP70, 18S and ITS-1 targets. We observed that the kDNA was the most sensitive but does not meet the criterion of exclusivity. The HSP70 presented a higher LoD in conventional PCR and qPCR in comparison with the other markers (1 × 101 and 1 × 10-1 equivalent parasites/mL respectively) and had a higher coefficient of variation in qPCR. No statistically significant differences were found between the days of the test with the four molecular markers. The present study revealed that the 18S marker presented the best performance in terms of analytical sensitivity and specificity for the qPCR in the species tested (species circulating in Colombia). Therefore, we recommend to explore the analytical and diagnostic performance in future studies using a broader number of species across America. PMID:29046670

  5. DNA polymerase preference determines PCR priming efficiency.

    Science.gov (United States)

    Pan, Wenjing; Byrne-Steele, Miranda; Wang, Chunlin; Lu, Stanley; Clemmons, Scott; Zahorchak, Robert J; Han, Jian

    2014-01-30

    Polymerase chain reaction (PCR) is one of the most important developments in modern biotechnology. However, PCR is known to introduce biases, especially during multiplex reactions. Recent studies have implicated the DNA polymerase as the primary source of bias, particularly initiation of polymerization on the template strand. In our study, amplification from a synthetic library containing a 12 nucleotide random portion was used to provide an in-depth characterization of DNA polymerase priming bias. The synthetic library was amplified with three commercially available DNA polymerases using an anchored primer with a random 3' hexamer end. After normalization, the next generation sequencing (NGS) results of the amplified libraries were directly compared to the unamplified synthetic library. Here, high throughput sequencing was used to systematically demonstrate and characterize DNA polymerase priming bias. We demonstrate that certain sequence motifs are preferred over others as primers where the six nucleotide sequences at the 3' end of the primer, as well as the sequences four base pairs downstream of the priming site, may influence priming efficiencies. DNA polymerases in the same family from two different commercial vendors prefer similar motifs, while another commercially available enzyme from a different DNA polymerase family prefers different motifs. Furthermore, the preferred priming motifs are GC-rich. The DNA polymerase preference for certain sequence motifs was verified by amplification from single-primer templates. We incorporated the observed DNA polymerase preference into a primer-design program that guides the placement of the primer to an optimal location on the template. DNA polymerase priming bias was characterized using a synthetic library amplification system and NGS. The characterization of DNA polymerase priming bias was then utilized to guide the primer-design process and demonstrate varying amplification efficiencies among three commercially

  6. Molecular quantification of environmental DNA using microfluidics and digital PCR.

    Science.gov (United States)

    Hoshino, Tatsuhiko; Inagaki, Fumio

    2012-09-01

    Real-time PCR has been widely used to evaluate gene abundance in natural microbial habitats. However, PCR-inhibitory substances often reduce the efficiency of PCR, leading to the underestimation of target gene copy numbers. Digital PCR using microfluidics is a new approach that allows absolute quantification of DNA molecules. In this study, digital PCR was applied to environmental samples, and the effect of PCR inhibitors on DNA quantification was tested. In the control experiment using λ DNA and humic acids, underestimation of λ DNA at 1/4400 of the theoretical value was observed with 6.58 ng μL(-1) humic acids. In contrast, digital PCR provided accurate quantification data with a concentration of humic acids up to 9.34 ng μL(-1). The inhibitory effect of paddy field soil extract on quantification of the archaeal 16S rRNA gene was also tested. By diluting the DNA extract, quantified copy numbers from real-time PCR and digital PCR became similar, indicating that dilution was a useful way to remedy PCR inhibition. The dilution strategy was, however, not applicable to all natural environmental samples. For example, when marine subsurface sediment samples were tested the copy number of archaeal 16S rRNA genes was 1.04×10(3) copies/g-sediment by digital PCR, whereas real-time PCR only resulted in 4.64×10(2) copies/g-sediment, which was most likely due to an inhibitory effect. The data from this study demonstrated that inhibitory substances had little effect on DNA quantification using microfluidics and digital PCR, and showed the great advantages of digital PCR in accurate quantifications of DNA extracted from various microbial habitats. Copyright © 2012 Elsevier GmbH. All rights reserved.

  7. Multiplex Amplification Refractory Mutation System PCR (ARMS-PCR) provides sequencing independent typing of canine parvovirus.

    Science.gov (United States)

    Chander, Vishal; Chakravarti, Soumendu; Gupta, Vikas; Nandi, Sukdeb; Singh, Mithilesh; Badasara, Surendra Kumar; Sharma, Chhavi; Mittal, Mitesh; Dandapat, S; Gupta, V K

    2016-12-01

    Canine parvovirus-2 antigenic variants (CPV-2a, CPV-2b and CPV-2c) ubiquitously distributed worldwide in canine population causes severe fatal gastroenteritis. Antigenic typing of CPV-2 remains a prime focus of research groups worldwide in understanding the disease epidemiology and virus evolution. The present study was thus envisioned to provide a simple sequencing independent, rapid, robust, specific, user-friendly technique for detecting and typing of presently circulating CPV-2 antigenic variants. ARMS-PCR strategy was employed using specific primers for CPV-2a, CPV-2b and CPV-2c to differentiate these antigenic types. ARMS-PCR was initially optimized with reference positive controls in two steps; where first reaction was used to differentiate CPV-2a from CPV-2b/CPV-2c. The second reaction was carried out with CPV-2c specific primers to confirm the presence of CPV-2c. Initial validation of the ARMS-PCR was carried out with 24 sequenced samples and the results were matched with the sequencing results. ARMS-PCR technique was further used to screen and type 90 suspected clinical samples. Randomly selected 15 suspected clinical samples that were typed with this technique were sequenced. The results of ARMS-PCR and the sequencing matched exactly with each other. The developed technique has a potential to become a sequencing independent method for simultaneous detection and typing of CPV-2 antigenic variants in veterinary disease diagnostic laboratories globally. Copyright © 2016 Elsevier B.V. All rights reserved.

  8. Casipoemas por Navidad : (1969-2010)

    OpenAIRE

    Sena Medina, Guillermo

    2011-01-01

    PRESENTACIÓN AL AIRE DE LA POESÍA RELIGIOSA Y NAVIDEÑA DE GUILLERMO SENA MEDINA Constituye un gran honor hacer la presentación de Guillermo Sena Medina por un doble motivo; por la larga y sincera amistad que nos une desde hace muchos años al socaire de nuestra común afición por la Historia, como cronistas oficiales de nuestros municipios; y, sobre todo y particularmente, por su rica y profunda personalidad como hombre de letras, nacida de su aún más rica y profunda personalidad humana. A...

  9. ReadqPCR and NormqPCR: R packages for the reading, quality checking and normalisation of RT-qPCR quantification cycle (Cq data

    Directory of Open Access Journals (Sweden)

    Perkins James R

    2012-07-01

    Full Text Available Abstract Background Measuring gene transcription using real-time reverse transcription polymerase chain reaction (RT-qPCR technology is a mainstay of molecular biology. Technologies now exist to measure the abundance of many transcripts in parallel. The selection of the optimal reference gene for the normalisation of this data is a recurring problem, and several algorithms have been developed in order to solve it. So far nothing in R exists to unite these methods, together with other functions to read in and normalise the data using the chosen reference gene(s. Results We have developed two R/Bioconductor packages, ReadqPCR and NormqPCR, intended for a user with some experience with high-throughput data analysis using R, who wishes to use R to analyse RT-qPCR data. We illustrate their potential use in a workflow analysing a generic RT-qPCR experiment, and apply this to a real dataset. Packages are available from http://www.bioconductor.org/packages/release/bioc/html/ReadqPCR.htmland http://www.bioconductor.org/packages/release/bioc/html/NormqPCR.html Conclusions These packages increase the repetoire of RT-qPCR analysis tools available to the R user and allow them to (amongst other things read their data into R, hold it in an ExpressionSet compatible R object, choose appropriate reference genes, normalise the data and look for differential expression between samples.

  10. Digital PCR as a tool to measure HIV persistence.

    Science.gov (United States)

    Rutsaert, Sofie; Bosman, Kobus; Trypsteen, Wim; Nijhuis, Monique; Vandekerckhove, Linos

    2018-01-30

    Although antiretroviral therapy is able to suppress HIV replication in infected patients, the virus persists and rebounds when treatment is stopped. In order to find a cure that can eradicate the latent reservoir, one must be able to quantify the persisting virus. Traditionally, HIV persistence studies have used real-time PCR (qPCR) to measure the viral reservoir represented by HIV DNA and RNA. Most recently, digital PCR is gaining popularity as a novel approach to nucleic acid quantification as it allows for absolute target quantification. Various commercial digital PCR platforms are nowadays available that implement the principle of digital PCR, of which Bio-Rad's QX200 ddPCR is currently the most used platform in HIV research. Quantification of HIV by digital PCR is proving to be a valuable improvement over qPCR as it is argued to have a higher robustness to mismatches between the primers-probe set and heterogeneous HIV, and forfeits the need for a standard curve, both of which are known to complicate reliable quantification. However, currently available digital PCR platforms occasionally struggle with unexplained false-positive partitions, and reliable segregation between positive and negative droplets remains disputed. Future developments and advancements of the digital PCR technology are promising to aid in the accurate quantification and characterization of the persistent HIV reservoir.

  11. Co-infection by porcine circovirus type 2 and porcine parvovirus in aborted fetuses and stillborn piglets in southern Brazil Co-infecção por circovírus suíno tipo 2 e parvo-vírus suíno em fetos abortados e natimortos suínos no Sul do Brasil

    Directory of Open Access Journals (Sweden)

    Caroline A. Pescador

    2007-10-01

    fetos mumificados de tamanhos variados. Dilatação ventricular, áreas pálidas miocárdicas e edema de mesocólon foram as lesões macroscópicas observadas. Escherichia coli co-infectou com PCV2 as amostras dos casos com edema de mesocólon. Lesões microscópicas incluíram miocardite não supurativa, necrose e fibrose miocárdicas, focos de mineralização e corpúsculos de inclusão em cardiomiócitos e pneumonia intersticial mononuclear. Entre os 121 fetos suínos abortados ou natimortos analisados, sete (5,78% tinham lesões compatíveis com origem viral e foram positivos pelas técnicas de imunoistoquímica e PCR para PCV2. Além disso, três (2.47% desses sete casos também foram confirmados como co-infectados com PPV através da PCR. Antígenos de PCV2 foram observados principalmente em macrógafos e no interior de miócitos dos fetos suínos abortados e natimortos. PCV2 e PPV foram detectados em diferentes estágios de gestação. PCV1 não foi associado isoladamente com feto ou natimorto afetado, mas estava presente em associação com PCV2 e/ou PPV em alguns desses produtos. Esses achados indicam que a infecção por PCV2, isoladamente ou em associação com PPV, deve ser considerada no diagnóstico de aborto infeccioso suíno no Brasil.

  12. Hematoquecia letal por angiostrongilosis abdominal

    Directory of Open Access Journals (Sweden)

    Gerzain Rodríguez

    2000-06-01

    Full Text Available Una niña indigena de 4 años de edad, procedente de Carurú, Vaupés, un municipio semiselvático de 2.000 habitantes, presentó hematoquecia repetida con sincope y muerte 4 días después, a pesar de recibir transfusiones sanguíneas. El estudio postmortem descubrió dos masas de 3-4 cm de diámetro en el ileon distal que, al microscopio, correspondieron a inflamación severa de toda la pared intestinal, rica en eosinofilos y en granulomas con células gigantes que circunscribian y fagocitaban huevos y larvas de Angiostrongylus costaricensis, helminto situado también dentro de las arterias de la submucosa, vasos que presentaban engrosamiento de la intima y focos con inflamación granulomatosa con eosinófilos. La niña no presentó leucocitosis ni eosinofilia. Este es el segundo caso de angiostrongilosis de esta región. El primero fue el de una mujer indigena de 34 años también con hematoquecia anemizante. La angiostrongilosis abdominal se diagnosticó por primera vez en Colombia en 1979, en un niño de 5 años, de Dagua, Valle. La niña aquí presentada es el sexto paciente con esta entidad en Colombia. La hematoquecia como manifestación predominante de la angiostrongilosis es extraordinaria para una enfermedad parasitaria; se ha registrado solamente en tres ocasiones anteriores. La abundancia de cristales de Charcot-Leyden, que se tiñen muy bien con la coloración de Ziehl-Neelsen o de Fite-Faraco, en los infiltrados de la pared intestinal. sugiere que su búsqueda en las heces puede ser una ayuda en el diagnóstico de casos sospechosos. pues, los huevos y las larvas no se eliminan en las heces y el diagnóstico definitivo de la angiostrongilosis es histopatológico. Revisamos las caracteristicas clinicas, parasitológicas e histopatológicas de esta parasitosis que será más prevalente en Colombia a medida que los médicos. especialmente los patólogos, la conozcan mejor.

  13. Pesquisa da prevalência do papilomavírus humano em amostras de tecido endometrial normal e com carcinoma pela técnica de PCR Search for human papillomavirus in samples of normal endometrial tissue and tissue with carcinoma by the PCR technique

    Directory of Open Access Journals (Sweden)

    Edison Natal Fedrizzi

    2004-05-01

    Full Text Available OBJETIVO: comparar a prevalência da presença do DNA do papilomavírus humano (HPV pela técnica de PCR em amostras de tecido endometrial normal e com carcinoma endometrial de mulheres submetidas a tratamento cirúrgico (histerectomia ou carcinoma endometrial e doença benigna. MÉTODOS: trata-se de um estudo observacional do tipo caso-controle onde foram avaliadas 100 mulheres (50 com endométrio normal e 50 com carcinoma endometrial quanto a presença do DNA do HPV em amostra tecidual conservada em blocos de parafina, pelo método de PCR. Foram excluídos os casos de carcinoma endometrial cujo sítio primário da lesão era duvidoso ou com história prévia ou atual de lesões pré-neoplásicas ou carcinoma do trato genital inferior. Variáveis como idade, tabagismo, trofismo endometrial, diferenciação escamosa e grau de diferenciação tumoral foram também avaliadas. RESULTADOS: o risco relativo estimado da presença do HPV foi o mesmo nas mulheres com e sem carcinoma endometrial. O HPV foi detectado em 8% dos casos de carcinoma e 10% no endométrio normal. Apesar de o HPV ter sido detectado 3,5 vezes mais em mulheres fumantes no grupo sem carcinoma, não houve diferença estatística. A presença do HPV também não esteve correlacionada com a idade das mulheres, trofismo endometrial, diferenciação escamosa e grau de diferenciação tumoral. Os HPV 16 e 18 (5 dos casos com o tipo 16 e 4 com o tipo 18 foram os vírus mais freqüentemente encontrados, tanto no tecido endometrial normal, quanto no carcinomatoso. Nenhum vírus de baixo risco oncogênico foi detectado nas amostras. CONCLUSÃO: o HPV está presente no tecido endometrial de mulheres com carcinoma endometrial na mesma proporção que nas com tecido endometrial normal, não se demonstrando a possível associação deste vírus no desenvolvimento do carcinoma endometrial.OBJECTIVE: to compare the prevalence of DNA of human papillomavirus (HPV, in samples of normal endometrial

  14. A new PCR approach for the identification of Fusarium graminearum Um novo protocolo de PCR para a identificação de Fusarium graminearum

    Directory of Open Access Journals (Sweden)

    Gleison Ricardo de Biazio

    2008-09-01

    cepas que foram morfologicamente e molecularmente identificadas como F. graminearum foram capazes de secretar a enzima e tiveram um resultado positivo no protocolo de PCR, utilizando os iniciadores direcionados para o gene gaoA. Nenhum fragmento de DNA foi amplificado quando foi utilizado o DNA genômico de várias outras espécies de Fusarium e de espécies de outros gêneros de fungos. Os resultados sugerem que o protocolo de PCR gerado é específico e pode ser considerado como uma nova ferramenta molecular para a identificação de cepas de F. graminearum. Além disso, o meio líquido formulado é uma alternativa barata para a avaliação da produção de GO por F. graminearum.

  15. COMPARISON OF 16S rRNA-PCR-RFLP, LipL32-PCR AND OmpL1-PCR METHODS IN THE DIAGNOSIS OF LEPTOSPIROSIS

    Directory of Open Access Journals (Sweden)

    Tülin GÜVEN GÖKMEN

    Full Text Available SUMMARY Leptospirosis is still one of the most important health problems in developing countries located in humid tropical and subtropical regions. Human infections are generally caused by exposure to water, soil or food contaminated with the urine of infected wild and domestic animals such as rodents and dogs. The clinical course of leptospirosis is variable and may be difficult to distinguish from many other infectious diseases. The dark-field microscopy (DFM, serology and nucleic acid amplification techniques are used to diagnose leptospirosis, however, a distinctive standard reference method is still lacking. Therefore, in this study, we aimed to determine the presence of Leptospira spp., to differentiate the pathogenic L. interrogans and the non-pathogenic L. biflexa, and also to determine the sensitivity and specificity values of molecular methods as an alternative to conventional ones. A total of 133 serum samples, from 47 humans and 86 cattle were evaluated by two conventional tests: the Microagglutination Test (MAT and the DFM, as well as three molecular methods, the 16S rRNA-PCR followed by Restriction Fragment Lenght Polymorphism (RFLP of the amplification products 16S rRNA-PCR-RFLP, LipL32-PCR and OmpL1-PCR. In this study, for L. interrogans, the specificity and sensitivity rates of the 16S rRNA-PCR and the LipL32-PCR were considered similar (100% versus 98.25% and 100% versus 98.68%, respectively. The OmpL1-PCR was able to classify L. interrogans into two intergroups, but this PCR was less sensitive (87.01% than the other two PCR methods. The 16S rRNA-PCR-RFLP could detect L. biflexa DNA, but LipL32-PCR and OmpL1-PCR could not. The 16S rRNA-PCR-RFLP provided an early and accurate diagnosis and was able to distinguish pathogenic and non-pathogenic Leptospira species, hence it may be used as an alternative method to the conventional gold standard techniques for the rapid disgnosis of leptospirosis.

  16. Typing of avian pathogenic Escherichia coli strains by REP-PCR Tipificação de amostras aviárias patogênicas de Escherichia coli pela REP-PCR

    Directory of Open Access Journals (Sweden)

    Marcelo Brocchi

    2006-06-01

    Full Text Available In the present study the repetitive extragenic palindromic (REP polymerase chain reaction (PCR technique was used to establish the clonal variability of 49 avian Escherichia coli (APEC strains isolated from different outbreak cases of septicemia (n=24, swollen head syndrome (n=14 and omphalitis (n=11. Thirty commensal strains isolated from poultry with no signs of these illnesses were used as control strains. The purified DNA of these strains produced electrophoretic profiles ranging from 0 to 15 bands with molecular sizes varying from 100 bp to 6.1 kb, allowing the grouping of the 79 strains into a dendrogram containing 49 REP-types. Although REP-PCR showed good discriminating power it was not able to group the strains either into specific pathogenic classes or to differentiate between pathogenic and non-pathogenic strains. On the contrary, we recently demonstrated that other techniques such as ERIC-PCR and isoenzyme profiles are appropriate to discriminate between commensal and APEC strains and also to group these strains into specific pathogenic classes. In conclusion, REP-PCR seems to be a technique neither efficient nor universal for APEC strains discrimination. However, the population clonal structure obtained with the use of REP-PCR must not be ignored particularly if one takes into account that the APEC pathogenic mechanisms are not completely understood yet.A técnica de REP (Repetitive extragenic palindrome-PCR foi utilizada para avaliar a variabilidade genética de 49 amostras de Escherichia coli patogênicas para aves (APEC, isoladas de aves de corte (frangos em diferentes surtos de septicemia (n=24, síndrome da cabeça inchada (n=14 e onfalite (n=11. Trinta amostras comensais, isoladas de frangos sem sinais de doença, foram utilizadas como controle. A análise do perfil eletroforético obtido por reação de REP-PCR utilizando DNA purificado das amostras evidenciou a amplificação de 0 a 15 bandas de DNA com pesos moleculares

  17. Encefalopatía por priones

    Directory of Open Access Journals (Sweden)

    Carlos Colegial

    1999-01-01

    Full Text Available Las encefalopatías espongiformes por priones son enfermedades neurodegenerativas que pueden ser esporádicas o transmisibles, ya sea por mecanismos infecciosos o hereditarios. Su investigación ha planteado enormes retos y en el recorrido histórico en busca de su causa dos médicos han recibido el premio Nobel de Medicina: Carleton Gajdusek, por sus trabajos en Nueva Guinea donde describió la transmisión infecciosa por ritos canibalísticos, que llevó a estudios de transmisión experimental en chimpancés y a su teoría de los "virus lentos" (por el largo período de incubación de la enfermedad.

  18. Multiplex enrichment quantitative PCR (ME-qPCR): a high-throughput, highly sensitive detection method for GMO identification.

    Science.gov (United States)

    Fu, Wei; Zhu, Pengyu; Wei, Shuang; Zhixin, Du; Wang, Chenguang; Wu, Xiyang; Li, Feiwu; Zhu, Shuifang

    2017-04-01

    Among all of the high-throughput detection methods, PCR-based methodologies are regarded as the most cost-efficient and feasible methodologies compared with the next-generation sequencing or ChIP-based methods. However, the PCR-based methods can only achieve multiplex detection up to 15-plex due to limitations imposed by the multiplex primer interactions. The detection throughput cannot meet the demands of high-throughput detection, such as SNP or gene expression analysis. Therefore, in our study, we have developed a new high-throughput PCR-based detection method, multiplex enrichment quantitative PCR (ME-qPCR), which is a combination of qPCR and nested PCR. The GMO content detection results in our study showed that ME-qPCR could achieve high-throughput detection up to 26-plex. Compared to the original qPCR, the Ct values of ME-qPCR were lower for the same group, which showed that ME-qPCR sensitivity is higher than the original qPCR. The absolute limit of detection for ME-qPCR could achieve levels as low as a single copy of the plant genome. Moreover, the specificity results showed that no cross-amplification occurred for irrelevant GMO events. After evaluation of all of the parameters, a practical evaluation was performed with different foods. The more stable amplification results, compared to qPCR, showed that ME-qPCR was suitable for GMO detection in foods. In conclusion, ME-qPCR achieved sensitive, high-throughput GMO detection in complex substrates, such as crops or food samples. In the future, ME-qPCR-based GMO content identification may positively impact SNP analysis or multiplex gene expression of food or agricultural samples. Graphical abstract For the first-step amplification, four primers (A, B, C, and D) have been added into the reaction volume. In this manner, four kinds of amplicons have been generated. All of these four amplicons could be regarded as the target of second-step PCR. For the second-step amplification, three parallels have been taken for

  19. Portable exhauster POR-007/Skid E and POR-008/Skid F storage plan

    International Nuclear Information System (INIS)

    Nelson, O.D.

    1998-01-01

    This document provides storage requirements for 1,000 CFM portable exhausters POR-O07/Skid E and POR-008/Skid F. These requirements are presented in three parts: preparation for storage, storage maintenance and testing, and retrieval from storage. The exhauster component identification numbers listed in this document contain the prefix POR-007 or POR-008 depending on which exhauster is being used

  20. Blood grouping based on PCR methods and agarose gel electrophoresis.

    Science.gov (United States)

    Sell, Ana Maria; Visentainer, Jeane Eliete Laguila

    2015-01-01

    The study of erythrocyte antigens continues to be an intense field of research, particularly after the development of molecular testing methods. More than 300 specificities have been described by the International Society for Blood Transfusion as belonging to 33 blood group systems. The polymerase chain reaction (PCR) is a central tool for red blood cells (RBC) genotyping. PCR and agarose gel electrophoresis are low cost, easy, and versatile in vitro methods for amplifying defined target DNA (RBC polymorphic region). Multiplex-PCR, AS-PCR (Specific Allele Polymerase Chain Reaction), and RFLP-PCR (Restriction Fragment Length Polymorphism-Polymerase Chain Reaction) techniques are usually to identify RBC polymorphisms. Furthermore, it is an easy methodology to implement. This chapter describes the PCR methodology and agarose gel electrophoresis to identify the polymorphisms of the Kell, Duffy, Kidd, and MNS blood group systems.

  1. Product differentiation during continuous-flow thermal gradient PCR.

    Science.gov (United States)

    Crews, Niel; Wittwer, Carl; Palais, Robert; Gale, Bruce

    2008-06-01

    A continuous-flow PCR microfluidic device was developed in which the target DNA product can be detected and identified during its amplification. This in situ characterization potentially eliminates the requirement for further post-PCR analysis. Multiple small targets have been amplified from human genomic DNA, having sizes of 108, 122, and 134 bp. With a DNA dye in the PCR mixture, the amplification and unique melting behavior of each sample is observed from a single fluorescent image. The melting behavior of the amplifying DNA, which depends on its molecular composition, occurs spatially in the thermal gradient PCR device, and can be observed with an optical resolution of 0.1 degrees C pixel(-1). Since many PCR cycles are within the field of view of the CCD camera, melting analysis can be performed at any cycle that contains a significant quantity of amplicon, thereby eliminating the cycle-selection challenges typically associated with continuous-flow PCR microfluidics.

  2. Selecting PCR for the Diagnosis of Intestinal Parasitosis

    DEFF Research Database (Denmark)

    Hartmeyer, G. N.; Hoegh, S. V.; Skov, M. N.

    2017-01-01

    Microscopy of stool samples is a labour-intensive and inaccurate technique for detection of intestinal parasites causing diarrhoea and replacement by PCR is attractive. Almost all cases of diarrhoea induced by parasites over a nine-year period in our laboratory were due to Giardia lamblia......, Cryptosporidium species, or Entamoeba histolytica detected by microscopy. We evaluated and selected in-house singleplex real-time PCR (RT-PCR) assays for these pathogens in 99 stool samples from patients suspected of having intestinal parasitosis tested by microscopy. The strategy included a genus-specific PCR...... assay for C. parvum and C. hominis, with subsequent identification by a PCR that distinguishes between the two species. G. lamblia was detected in five and C. parvum in one out of 68 microscopy-negative samples. The performance of the in-house RT-PCR assays was compared to three commercially available...

  3. Standardization of diagnostic PCR for the detection of foodborne pathogens

    DEFF Research Database (Denmark)

    Malorny, B.; Tassios, P.T.; Radstrom, P.

    2003-01-01

    In vitro amplification of nucleic acids using the polymerase chain reaction (PCR) has become, since its discovery in the 1980s, a powerful diagnostic tool for the analysis of microbial infections as well as for the analysis of microorganisms in food samples. However, despite its potential, PCR has...... neither gained wide acceptance in routine diagnostics nor been widely incorporated in standardized methods. Lack of validation and standard protocols, as well as variable quality of reagents and equipment, influence the efficient dissemination of PCR methodology from expert research laboratories to end......-user laboratories. Moreover, the food industry understandably requires and expects officially approved standards. Recognizing this, in 1999, the European Commission approved the research project, FOOD-PCR (http://www.PCR.dk), which aims to validate and standardize the use of diagnostic PCR for the detection...

  4. [Retrospective study of the implementation of the qualitative PCR technique in biological samples for monitoring toxoplasmosis in pediatric patients receiving hematopoietic stem cell transplantation].

    Science.gov (United States)

    Nigro, Mónica G; Figueroa, Carlos; Ledesma, Bibiana A

    2014-01-01

    Toxoplasmosis is an opportunistic infection caused by the parasite Toxoplasma gondii. The infection is severe and difficult to diagnose in patients receiving allogeneic hematopoietic stem cell transplantation (HSCT). Twelve patients receiving HSCT were monitored post-transplant, by qualitative PCR at the Children's Hospital S.A.M.I.C. "Prof. Dr. Juan P. Garrahan". The monitoring of these patients was defined by a history of positive serology for toxoplasmosis in the donor or recipient and because their hematologic condition did not allow the use of trimethoprim-sulfamethoxazole for prophylaxis. During the patients' monitoring, two of them with positive PCR results showed signs of illness by T. gondii and were treated with pyrimethamine-clindamycin. In two other patients, toxoplasmosis was the cause of death and an autopsy finding, showing negative PCR results. Four patients without clinical manifestations received treatment for toxoplasmosis because of positive PCR detection. In four patients there were no signs of toxoplasmosis disease and negative PCR results during follow-up. The qualitative PCR technique proved useful for the detection of toxoplasmosis reactivation in HSCT recipients, but has limitations in monitoring and making clinical decisions due to the persistence of positive PCR over time and manifestations of toxicity caused by the treatment. Copyright © 2014 Asociación Argentina de Microbiología. Publicado por Elsevier España. All rights reserved.

  5. Comparison of multiplex reverse transcription-PCR-enzyme ...

    African Journals Online (AJOL)

    Comparison of multiplex reverse transcription-PCR-enzyme hybridization assay with immunofluorescence techniques for the detection of four viral respiratory pathogens in pediatric community acquired pneumonia.

  6. Comparative evaluation of conventional RT-PCR and real-time RT-PCR (RRT-PCR) for detection of avian metapneumovirus subtype A

    OpenAIRE

    Ferreira, HL; Spilki, FR; dos Santos, MMAB; de Almeida, RS; Arns, CW

    2009-01-01

    Avian metapneumovirus (AMPV) belongs to Metapneumovirus genus of Paramyxoviridae family. Virus isolation, serology, and detection of genomic RNA are used as diagnostic methods for AMPV. The aim of the present study was to compare the detection of six subgroup A AMPV isolates (AMPV/A) viral RNA by using different conventional and real time RT-PCR methods. Two new RT-PCR tests and two real time RT-PCR tests, both detecting fusion (F) gene and nucleocapsid (N) gene were compared with an establis...

  7. Análise do uso da terra na microbacia do Arroio do Meio - Santa Maria - RS, por Sistema de Informações Geográficas e imagem de satélite

    Directory of Open Access Journals (Sweden)

    Piroli Edson Luís

    2002-01-01

    Full Text Available O objetivo desse trabalho foi verificar a viabilidade do uso de um Sistema de Informações Geográficas (SIG e a imagem de satélite para a análise do uso atual da terra e localização de áreas onde possam estar ocorrendo conflitos entre capacidade e uso do solo, na microbacia hidrográfica do Arroio do Meio. Foram utilizadas técnicas de geoprocessamento, como álgebra entre mapas, consulta ao banco de dados e reclassificação de imagens. Uma microbacia foi escolhida como objeto deste estudo, por ser considerada por muitos autores como sendo uma das melhores unidades para o planejamento e desenvolvimento sócioeconômico dos habitantes do meio rural. Na microbacia estudada, foram encontrados 555ha cobertos com florestas, compreendendo 24% da área total. As lavouras com área de 1.314ha ocupam a maior parte da microbacia (56%. Os campos de pastagens cobrem 184ha, ou seja, 8% da área total. As áreas alagadas representam 11% da área da microbacia, tendo respectivamente 265ha. Foram detectados ainda, 31ha sombreados (1% onde não se determinou com exatidão o uso da terra. Nas áreas com declividade superior a 47%, foram detectados 32ha sem cobertura de florestas, perfazendo 1,4% da área da microbacia. Em declives superiores a 30%, existem 71ha (3% sendo usados para a agricultura. A área ocupada com Chernossolos e Neossolos Litólicos, unidade de mapeamento Ciríaco-Charrua em declividade maior que 30% sem cobertura florestal é de 14ha (0,6%. De acordo com a declividade e o solo, as áreas de conflito alcançam 5% da área total, o que demonstra que, na maior parte da microbacia, a terra está sendo usada de acordo com sua capacidade.

  8. Dinámica folicular en yeguas paso fino colombiano medido por ultrasonografía en la Sabana de Bogotá

    Directory of Open Access Journals (Sweden)

    Germán Ramírez

    2010-06-01

    Full Text Available Este estudio surgió como medio para obtener un parámetro de crecimiento diario folicular, momento de la ovulación, y determinar cuál es el ovario con mayor número de ovulaciones en la yegua de Paso Fino Colombiano con el fin de aportar algunos datos de dinámica folicular en la yegua de esta especie, ya que la información disponible en la literatura data generalmente en otras razas y condiciones diferentes al trópico. Para el desarrollo de este proyecto se tomaron como muestra cincuenta (50 yeguas ubicadas en la sabana de Bogotá: (Chía – Cundinamarca, a una altura de 2.652 metros sobre el nivel del mar, temperatura promedio de 12° C y pluviosidad 1.500 mm; la edad de los animales osciló entre los cinco y diez años. Se realizaron ecografías periódicas día por medio por palpación rectal, con el fin de realizar un seguimiento del crecimiento folicular una vez detectado un folículo dominante (>30 mm de diámetro, hasta su ovulación. Los datos fueron analizados por medio de estadística descriptiva, con desviación estándar, además de una prueba F para varianzas desiguales e iguales para determinar diferencias en el tamaño folicular a la ovulación y crecimiento diario folicular entre el ovario derecho e izquierdo. Teniendo como resultado que la yegua de paso fino colombiano tiene un crecimiento diario folicular de 2,04+/-0,63 mm, un tamaño folicular a la ovulación de 41,34+/-2,14 mm, y que de los cincuenta ciclos estrales analizados el 60% fue por el ovario izquierdo y el 40% restante por el ovario derecho. Las diferencias en cuanto a tamaño folicular a la ovulación no fueron significativas (P>0,50 entre los ovarios, mientras que para el crecimiento diario folicular sí hubo diferencia significativa (P<0,02 lo que significa que los folículos del ovario derecho tuvieron un crecimiento mayor a los del ovario izquierdo. Con estos resultados se obtuvo que la yegua de paso fino colombiano se comporta reproductivamente de

  9. Comparison of kDNA PCR-hybridization assay with three PCR methods for canines visceral Leishmaniasis diagnosis

    Energy Technology Data Exchange (ETDEWEB)

    Pilatti, Marcia M.; Andrade, Antero S.R. [Centro de Desenvolvimento da Tecnologia Nuclear (CDTN/CNEN-MG), Belo Horizonte, MG (Brazil)], e-mail: marciapilatti@yahoo.com.br, e-mail: antero@cdtn.br; Ferreira, Sidney A. [Universidade Federal de Minas Gerais (UFMG), Belo Horizonte, MG (Brazil). Dept. de Parasitologia], e-mail: saninoalmeida@gmail.com

    2009-07-01

    The sensitivity of the kDNA PCR-Hybridization assay, which uses radioactive DNA probes (labeled with {sup 32}P), was compared with three conventional PCR methods used for canine visceral leishmaniasis diagnosis. All PCR methods had two steps: a first amplification followed by hybridization or by a new amplification (nested or semi nested). Two methods (kDNA PCR-Hybridization and kDNA snPCR) used primers addressed to kinetoplast minicircles and the other two methods to the coding (LnPCR) and intergenic noncoding regions (ITS-1 nPCR) of the ribosomal rRNA genes. The comparison was accomplished in two groups of 23 infected dogs using samples collected by the conjunctival swab procedure. In the Group 1 the DNA was extracted from cotton swabs by phenol-chloroform and in Group 2 by boiling. The most efficient PCR methods in the Group 1 were those based on kDNA targets. The kDNA PCR-Hybridization was able to detect parasites in 22/23 dogs (95.6%) and in 40/46 samples (86.9%). The kDNA snPCR was positive for 21/23 dogs (91.3%) and for 40/46 samples (86.9%). The positivities of the kDNA based methods were significantly higher than the positivities verified for the methods based on ribosomal rRNA genes (p<0.05). In the Group 2 the kDNA PCR- Hybridization showed a better performance detecting parasites in 18/23 dogs (78.3%) and in 31/46 samples (67.4%), significantly higher than the other three methods (p<0.05). The higher sensitivity of the minicircle kDNA based assays reported by others was confirmed in this study and kDNA PCR-Hybridization showed the best sensitivity among the assays evaluated. (author)

  10. Comparison of kDNA PCR-hybridization assay with three PCR methods for canines visceral Leishmaniasis diagnosis

    International Nuclear Information System (INIS)

    Pilatti, Marcia M.; Andrade, Antero S.R.; Ferreira, Sidney A.

    2009-01-01

    The sensitivity of the kDNA PCR-Hybridization assay, which uses radioactive DNA probes (labeled with 32 P), was compared with three conventional PCR methods used for canine visceral leishmaniasis diagnosis. All PCR methods had two steps: a first amplification followed by hybridization or by a new amplification (nested or semi nested). Two methods (kDNA PCR-Hybridization and kDNA snPCR) used primers addressed to kinetoplast minicircles and the other two methods to the coding (LnPCR) and intergenic noncoding regions (ITS-1 nPCR) of the ribosomal rRNA genes. The comparison was accomplished in two groups of 23 infected dogs using samples collected by the conjunctival swab procedure. In the Group 1 the DNA was extracted from cotton swabs by phenol-chloroform and in Group 2 by boiling. The most efficient PCR methods in the Group 1 were those based on kDNA targets. The kDNA PCR-Hybridization was able to detect parasites in 22/23 dogs (95.6%) and in 40/46 samples (86.9%). The kDNA snPCR was positive for 21/23 dogs (91.3%) and for 40/46 samples (86.9%). The positivities of the kDNA based methods were significantly higher than the positivities verified for the methods based on ribosomal rRNA genes (p<0.05). In the Group 2 the kDNA PCR- Hybridization showed a better performance detecting parasites in 18/23 dogs (78.3%) and in 31/46 samples (67.4%), significantly higher than the other three methods (p<0.05). The higher sensitivity of the minicircle kDNA based assays reported by others was confirmed in this study and kDNA PCR-Hybridization showed the best sensitivity among the assays evaluated. (author)

  11. Meningoencefalite por Listeria monocytogenes em ovinos Meningoencephalitis in sheep caused by Listeria monocytogenes

    Directory of Open Access Journals (Sweden)

    Daniel R. Rissi

    2010-01-01

    Full Text Available São descritos sete casos de doença neurológica em ovinos por Listeria monocytogenes no Rio Grande do Sul e Paraná entre 2000 e 2007. Foram afetados ovinos com idades entre 12-24 meses. Os casos ocorreram no verão e início da primavera e os índices gerais de morbidade e letalidade foram de 3,15% e 100%, respectivamente. Quando essa informação estava disponível, nenhum dos ovinos afetados era alimentado com silagem. Em três propriedades havia contato próximo dos ovinos afetados com outras espécies. A evolução do quadro clínico foi de 12 horas a três dias e os sinais clínicos foram caracterizados por decúbito (7/7, desvio da cabeça (4/7, incoordenação (3/7, depressão (3/7, andar em círculos (2/7, cegueira unilateral, emagrecimento progressivo, febre, midríase, movimentos de pedalagem, nistagmo lateral, opistótono, paralisia flácida dos membros pélvicos ou dos quatro membros, salivação excessiva e tremores (1/7 cada. Histologicamente observou-se encefalite com microabscessos, predominantemente unilateral com variáveis graus de gliose e alterações degenerativas como esferóides axonais e infiltração de células Gitter. As lesões se estendiam desde a medula oblonga até o mesencéfalo. Antígenos de Listeria monocytogenes foram detectados por imuno-histoquímica em seções de tronco encefálico de todos os ovinos afetados. O diagnóstico foi realizado com base nos achados epidemiológicos e clinico-patológicos, e confirmado pela imuno-histoquímica (IHQ utilizando anticorpo policlonal anti-L. monocytogenes.Seven cases of neurological disease in sheep caused by Listeria monocytogenes in Rio Grande do Sul and Paraná state, southern Brazil are described. The cases occurred between 2000 and 2007 and 12-24-month-old sheep were affected. Overall morbidity and lethality rates were 3.15% and 100%, respectively. Cases occurred in the summer and early spring. When this information was available, affected sheep had not been

  12. Verdad por definición

    OpenAIRE

    Garrido Garrido, Julián

    1997-01-01

    Not available.La verdad por definición es un tipo peculiar de verdad científica, distinguible de las verdades lógicas, matemáticas y empíricas. La definición, por su parte, designa diversos procedimientos de asignación de significado, cuyas diferencias exigen una adjetivación cuidadosa: definiciones ostensivas y operacionales, definiciones de diccionario y definiciones teóricas. Pero sólo las del último tipo son verdaderas por definición. En el presente artículo se precisa el concepto formal ...

  13. Síndrome de Munchausen por mandato

    OpenAIRE

    Esteban, Miguel A.; Pérez, Miriam R.; Bracco, Anahí

    2006-01-01

    El Síndrome de Munchausen por mandato, "un trastorno ficticio, por el cual la enfermedad del niño es inducida, promovida o provocada por la persona más próxima a él, generalmente su madre", es todavía mal conocido y su génesis imperfectamente comprendida. Esta comunicación está destinada a esclarecer al pediatra esta patología, con elevada morbilidad y secuelas, como así también de altísima mo...

  14. Study On Application Of Molecular Techniques (RAPD-PCR And RAMP-PCR) To Detect Mutation In Rice Breeding

    International Nuclear Information System (INIS)

    Hoang Thi My Linh; Phan, D. T. Son; Nguyen Thi Vang; Nguyen, T. T. Hien; Le XuanTham

    2007-01-01

    The project was carried out in 2007 with the purpose of consideration for using the two simple and inexpensive molecular techniques to estimate changes in DNA of rice mutant after gamma irradiation. Three rice cultivars: Basmati370, Tam Thom (TT1), IR64 and three gamma irradiated mutants BDS, TDS and VND 95-20 respectively, were used. Suitable DNA extraction procedure was obtained. PCR optimization was conducted on three important factors including: amount of MgCl 2 , DNA concentration and annealing temperature. 2.5 mM of MgCl 2 for RAPD-PCR and 3.75 mM for RAMP-PCR were found the best. 40 ng DNA provided a good amplification for RAMP-PCR; this figure was 50 ng for RAPD-PCR. Annealing temperatures were determined at 36 o C for RAPD primer and at 55±3 o C for Microsatellite primer. Final results showed that, both RAPD-PCR and RAMP-PCR could detect changes in DNA of rice mutants after gamma irradiation compared to their parents. Percentage of DNA changes determined by RAPD-PCR and RAMP-PCR on Basmati370 and its mutant BDS were 11.49% and 21.2% respectively; These on TT1 and TDS were 8.98% and 15.4%; and on IR64 and VND 95-20 were 3.45% and 4.95%. (author)

  15. Comparison of ELISA, nested PCR and sequencing and a novel qPCR for detection of Giardia isolates from Jordan.

    Science.gov (United States)

    Hijjawi, Nawal; Yang, Rongchang; Hatmal, Ma'mon; Yassin, Yasmeen; Mharib, Taghrid; Mukbel, Rami; Mahmoud, Sameer Alhaj; Al-Shudifat, Abdel-Ellah; Ryan, Una

    2018-02-01

    Little is known about the prevalence of Giardia duodenalis in human patients in Jordan and all previous studies have used direct microscopy, which lacks sensitivity. The present study developed a novel quantitative PCR (qPCR) assay at the β-giardin (bg) locus and evaluated its use as a frontline test for the diagnosis of giardiasis in comparison with a commercially available ELISA using nested PCR and sequencing of the glutamate dehydrogenase (gdh) locus (gdh nPCR) as the gold standard. A total of 96 human faecal samples were collected from 96 patients suffering from diarrhoea from 5 regions of Jordan and were screened using the ELISA and qPCR. The analytical specificity of the bg qPCR assay revealed no cross-reactions with other genera and detected all the Giardia isolates tested. Analytical sensitivity was 1 Giardia cyst per μl of DNA extract. The overall prevalence of Giardia was 64.6%. The clinical sensitivity and specificity of the bg qPCR was 89.9% and 82.9% respectively compared to 76.5 and 68.0% for the ELISA. This study is the first to compare three different methods (ELISA, bg qPCR, nested PCR and sequencing at the gdh locus) to diagnose Jordanian patients suffering from giardiasis and to analyze their demographic data. Copyright © 2018 Elsevier Inc. All rights reserved.

  16. Metais pesados, densidade e atividade microbiana em solo contaminado por rejeitos de indústria de zinco

    Directory of Open Access Journals (Sweden)

    H. E. Dias-Júnior

    1998-12-01

    Full Text Available As atividades relacionadas com mineração e indústria metalúrgica são responsáveis pela poluição de extensas áreas de solo em todo o mundo, sendo seus efeitos ainda pouco conhecidos nas condições brasileiras. No presente trabalho, os teores totais e solúveis em água de metais pesados, a densidade e a atividade microbiana foram avaliados em 1996, em sete locais de uma área de deposição de rejeitos da industrialização de zinco, em elevado estádio de degradação, e num local fora da área contaminada, considerado como referência, no estado de Minas Gerais. Todos os locais contaminados apresentaram elevados teores de metais pesados totais e solúveis em água, atingindo, respectivamente, os seguintes valores máximos, em mg kg-1: 11.969 e 726 de Zn; 109 e 18 de Cd; 1.016 e 0 de Pb e 887 e 8 de Cu. Todas as características biológicas avaliadas foram afetadas pelos elevados teores de metais com exceção do número de amonificadores. O C-biomassa apresentou redução acima de 80% em quatro locais contaminados em relação ao local fora da área contaminada. A respiração basal do solo foi maior no local contaminado coberto com Andropogon sp. e menor nos sítios sem vegetação. O número de actinomicetos e de fungos cultiváveis foi menos afetado pela contaminação que o número total de bactérias. Azospirillum spp. foram detectados apenas no local de referência. Oxidantes de amônio foram verificados em somente dois locais que continham vegetação, enquanto os oxidantes de nitrito foram detectados na maioria dos locais amostrados. Verificou-se a tendência de maior densidade e atividade microbiana nos locais contaminados e com algum tipo de vegetação. A concentração total e solúvel de Zn, de Cd e de Cu correlacionou-se fracamente com as características biológicas, exceto para o qCO2, o qual se mostrou promissor como indicador da contaminação com esses metais. Os dados indicaram uma interação qu

  17. La pintura vista por un pintor joven

    Directory of Open Access Journals (Sweden)

    Fuentes Pozo, Pedro

    2000-01-01

    Full Text Available Not available

    El pintor se define a lo largo del tiempo como un artista en mutación influenciado por la sociedad de su momento y por su propio mundo interior Esto ha contribuido a la creación de distintos estilos pictóricos y a una evolución que conlleva la despreocupación por el aspecto formal en aras de una introspección al mundo interior En el cambio constante se refleja la búsqueda del artista que valida la intemporalidad del arte. Así, el verdadero artista sobrevive al aplauso o rechazo de la crítica, convirtiéndose en un verdadero hombre renancentista preocupado por el hallazgo del conocimiento universal.

  18. 14 Nobel, preocupados por el CERN

    CERN Multimedia

    Rivera, A

    2003-01-01

    "E l presidente del Consejo del CERN (Laboratorio Europeo de Fisica de Particulas, junto a Ginebra), Maurice Bourquin, ha recibido una carta firmada por un grupo de cientificos muy especiales: 14 premios Nobel de Fisica" (1 page).

  19. Human fecal source identification with real-time quantitative PCR

    Science.gov (United States)

    Waterborne diseases represent a significant public health risk worldwide, and can originate from contact with water contaminated with human fecal material. We describe a real-time quantitative PCR (qPCR) method that targets a Bacteroides dori human-associated genetic marker for...

  20. Comparison between ICT and PCR for diagnosis of Chlamydia trachomatis.

    Science.gov (United States)

    Khan, E R; Hossain, M A; Paul, S K; Mahmud, C; Hasan, M M; Rahman, M M; Nahar, K; Kubayashi, N

    2012-04-01

    Chlamydia trachomatis is an obligate intracellular gram-negative bacterium which is the most prevalent cause of bacterial sexually transmitted infections (STI). The present study was carried to diagnose genital Chlamydia trachomatis infection among women of reproductive age, attending Mymensingh Medical College Hospital, during July 2009 to June 2010 by Immunochromatographic test (ICT) and Polymerase chain reaction (PCR). A total of 70 females were included in this study. Out of 70 cases 56 were symptomatic and 14 asymptomatic. Endocervical swabs were collected from each of the cases and examined by Immunochromatographic test (ICT) for antigen detection and Polymerase chain reaction (PCR) for detection of endogenous plasmid-based nucleic acid. A total 29(41.4%) of the cases were found positive for C. trachomatis either by ICT or PCR. Of the 56 symptomatic cases, 19(33.9%) were found ICT positive and 17(30.4%) were PCR positive. Among 14 asymptomatic females, 2(14.3%) were ICT positive and none were PCR positive. Though PCR is highly sensitive but a total of twelve cases were found ICT positive but PCR negative. It may be due to presence of plasmid deficient strain of C trachomatis which could be amplified by ompA based (Chromosomal gene) multiplex PCR.

  1. Comparison of Nested-PCR technique and culture method in ...

    African Journals Online (AJOL)

    USER

    2010-04-05

    Apr 5, 2010 ... Full Length Research Paper. Comparison of ... The aim of the present study was to evaluate the diagnostic value of nested PCR in genitourinary ... method. Based on obtained results, the positivity rate of urine samples in this study was 5.0% by using culture and PCR methods and 2.5% for acid fast staining.

  2. Development of a competitive PCR assay for the quantification of ...

    African Journals Online (AJOL)

    The E. coli malate dehydrogenase Mdh house-keeping gene was modified and used as an internal control and competitor DNA for the c-PCR. E. coli cell concentration equivalents ranging from 20 to 2 x 104 cells ml-1 could be quantified with the c-PCR. Fifty-three water samples from various sources were tested with the ...

  3. Evaluation of PCR for diagnosis of visceral leishmaniasis

    NARCIS (Netherlands)

    Osman, O. F.; Oskam, L.; Zijlstra, E. E.; Kroon, N. C.; Schoone, G. J.; Khalil, E. T.; El-Hassan, A. M.; Kager, P. A.

    1997-01-01

    An evaluation of Leishmania PCR was performed with bone marrow, lymph node, and blood samples from 492 patients, 60 positive controls, and 90 negative controls. Results were compared with microscopy results for Giemsa-stained smears. PCR and microscopy of lymph node and bone marrow aspirates from

  4. Digital PCR for direct quantification of viruses without DNA extraction.

    Science.gov (United States)

    Pavšič, Jernej; Žel, Jana; Milavec, Mojca

    2016-01-01

    DNA extraction before amplification is considered an essential step for quantification of viral DNA using real-time PCR (qPCR). However, this can directly affect the final measurements due to variable DNA yields and removal of inhibitors, which leads to increased inter-laboratory variability of qPCR measurements and reduced agreement on viral loads. Digital PCR (dPCR) might be an advantageous methodology for the measurement of virus concentrations, as it does not depend on any calibration material and it has higher tolerance to inhibitors. DNA quantification without an extraction step (i.e. direct quantification) was performed here using dPCR and two different human cytomegalovirus whole-virus materials. Two dPCR platforms were used for this direct quantification of the viral DNA, and these were compared with quantification of the extracted viral DNA in terms of yield and variability. Direct quantification of both whole-virus materials present in simple matrices like cell lysate or Tris-HCl buffer provided repeatable measurements of virus concentrations that were probably in closer agreement with the actual viral load than when estimated through quantification of the extracted DNA. Direct dPCR quantification of other viruses, reference materials and clinically relevant matrices is now needed to show the full versatility of this very promising and cost-efficient development in virus quantification.

  5. Inhibitory effect of common microfluidic materials on PCR outcome

    KAUST Repository

    Kodzius, Rimantas

    2013-10-10

    In this study, we established a simple method for evaluating the PCR compatibility of various common materials employed when fabricating microfluidic chips, including silicon, several kinds of silicon oxide, glasses, plastics, wax, and adhesives. Two-temperature PCR was performed with these materials to determine their PCR-inhibitory effect. In most cases, adding bovine serum albumin effectively improved the reaction yield. We also studied the individual PCR components from the standpoint of adsorption. Most of the materials did not inhibit the DNA, although they noticeably interacted with the polymerase. We provide a simple method of performing PCR-compatibility testing of materials using inexpensive instrumentation that is common in molecular biology laboratories. Furthermore, our method is direct, being performed under actual PCR conditions with high temperature. Our results provide an overview of materials that are PCR-friendly for fabricating microfluidic devices. The PCR reaction, without any additives, performed best with pyrex glass, and it performed worst with PMMA or acrylic glue materials.

  6. Detection of Bacillus spores using PCR and FTA filters.

    Science.gov (United States)

    Lampel, Keith A; Dyer, Deanne; Kornegay, Leroy; Orlandi, Palmer A

    2004-05-01

    Emphasis has been placed on developing and implementing rapid detection systems for microbial pathogens. We have explored the utility of expanding FTA filter technology for the preparation of template DNA for PCR from bacterial spores. Isolated spores from several Bacillus spp., B. subtilis, B. cereus, and B. megaterium, were applied to FTA filters, and specific DNA products were amplified by PCR. Spore preparations were examined microscopically to ensure that the presence of vegetative cells, if any, did not yield misleading results. PCR primers SRM86 and SRM87 targeted a conserved region of bacterial rRNA genes, whereas primers Bsub5F and Bsub3R amplified a product from a conserved sequence of the B. subtilis rRNA gene. With the use of the latter set of primers for nested PCR, the sensitivity of the PCR-based assay was increased. Overall, 53 spores could be detected after the first round of PCR, and the sensitivity was increased to five spores by nested PCR. FTA filters are an excellent platform to remove PCR inhibitors and have universal applications for environmental, clinical, and food samples.

  7. Inhibitory effect of common microfluidic materials on PCR outcome

    KAUST Repository

    Kodzius, Rimantas; Xiao, Kang; Wu, Jinbo; Yi, Xin; Gong, Xiuqing; Foulds, Ian G.; Wen, Weijia

    2013-01-01

    In this study, we established a simple method for evaluating the PCR compatibility of various common materials employed when fabricating microfluidic chips, including silicon, several kinds of silicon oxide, glasses, plastics, wax, and adhesives. Two-temperature PCR was performed with these materials to determine their PCR-inhibitory effect. In most cases, adding bovine serum albumin effectively improved the reaction yield. We also studied the individual PCR components from the standpoint of adsorption. Most of the materials did not inhibit the DNA, although they noticeably interacted with the polymerase. We provide a simple method of performing PCR-compatibility testing of materials using inexpensive instrumentation that is common in molecular biology laboratories. Furthermore, our method is direct, being performed under actual PCR conditions with high temperature. Our results provide an overview of materials that are PCR-friendly for fabricating microfluidic devices. The PCR reaction, without any additives, performed best with pyrex glass, and it performed worst with PMMA or acrylic glue materials.

  8. Splinkerette PCR for mapping transposable elements in Drosophila.

    Directory of Open Access Journals (Sweden)

    Christopher J Potter

    2010-04-01

    Full Text Available Transposable elements (such as the P-element and piggyBac have been used to introduce thousands of transgenic constructs into the Drosophila genome. These transgenic constructs serve many roles, from assaying gene/cell function, to controlling chromosome arm rearrangement. Knowing the precise genomic insertion site for the transposable element is often desired. This enables identification of genomic enhancer regions trapped by an enhancer trap, identification of the gene mutated by a transposon insertion, or simplifying recombination experiments. The most commonly used transgene mapping method is inverse PCR (iPCR. Although usually effective, limitations with iPCR hinder its ability to isolate flanking genomic DNA in complex genomic loci, such as those that contain natural transposons. Here we report the adaptation of the splinkerette PCR (spPCR method for the isolation of flanking genomic DNA of any P-element or piggyBac. We report a simple and detailed protocol for spPCR. We use spPCR to 1 map a GAL4 enhancer trap located inside a natural transposon, pinpointing a master regulatory region for olfactory neuron expression in the brain; and 2 map all commonly used centromeric FRT insertion sites. The ease, efficiency, and efficacy of spPCR could make it a favored choice for the mapping of transposable element in Drosophila.

  9. Micetoma por Nocardia brasiliensis: reporte de caso

    Directory of Open Access Journals (Sweden)

    Miriam Guevara R

    2003-07-01

    Full Text Available Se presenta el caso de un paciente peruano, agricultor, con una infección cutánea de origen traumático causada por Nocardia brasiliensis, que evolucionó hacia la amputación del miembro inferior afectado. El diagnóstico se realizó por examen directo y cultivo del espécimen.

  10. Biorremediación de arsénico mediada por microorganismos genéticamente modificados

    Directory of Open Access Journals (Sweden)

    Her Lizeth Rodríguez Martínez

    2017-10-01

    Full Text Available Las actividades antropogénicas aumentan la movilización y distribución de los metales pesados, si se rebasan los estándares permitidos por la normativa internacional resultan un serio problema para la salud de los seres vivos y el medio ambiente. Uno de los casos más alarmantes es el del arsénico; su presencia en agua potable y suelos (y por ende en alimentos es tal que hay reportados cientos de casos de intoxicación severa en países como China y Bangladesh. Debido a las actividades mineras, en algunas zonas de México como el Estado de Chihuahua, también se han encontrado altas concentraciones del elemento. Por el peligro que representa, la necesidad de encontrar alternativas para la remediación de sitios contaminados con arsénico es de suma importancia. Si bien existen diversos métodos físicos y químicos que se han usado desde hace décadas, la biorremediación constituye una alternativa prometedora que ofrece ventajas económicas y es eficiente. Esto último se debe a que se han aislado microorganismos que pueden resistir altas concentraciones de arsénico e incorporarlo a procesos metabólicos específicos gracias a mecanismos como la enzima arsenito oxidasa (AOX. Aún cuando el sistema de oxidación del arsénico no se ha descifrado completamente ha sido posible identificarlo en un gran número de microorganismos a través de técnicas de ingeniería genética, mismas que de manera reciente se han utilizado para potencializar la capacidad de las cepas silvestres. El objetivo de este documento consistió en hacer una revisión general sobre la biorremediación del arsénico y algunas estrategias como la detoxificación de arsénico (III por medio de AOX con el f in de encontrar una solución factible al problema detectado en el estado de Chihuahua. Tras la vasta cantidad de información recabada se determinó que la ingeniería genética resulta una herramienta prometedora para lograr la biorremediación de los metales

  11. Multiplex PCR identification of Taenia spp. in rodents and carnivores.

    Science.gov (United States)

    Al-Sabi, Mohammad N S; Kapel, Christian M O

    2011-11-01

    The genus Taenia includes several species of veterinary and public health importance, but diagnosis of the etiological agent in definitive and intermediate hosts often relies on labor intensive and few specific morphometric criteria, especially in immature worms and underdeveloped metacestodes. In the present study, a multiplex PCR, based on five primers targeting the 18S rDNA and ITS2 sequences, produced a species-specific banding patterns for a range of Taenia spp. Species typing by the multiplex PCR was compared to morphological identification and sequencing of cox1 and/or 12S rDNA genes. As compared to sequencing, the multiplex PCR identified 31 of 32 Taenia metacestodes from rodents, whereas only 14 cysts were specifically identified by morphology. Likewise, the multiplex PCR identified 108 of 130 adult worms, while only 57 were identified to species by morphology. The tested multiplex PCR system may potentially be used for studies of Taenia spp. transmitted between rodents and carnivores.

  12. Tatuaje por amalgama. Reporte de un caso

    Directory of Open Access Journals (Sweden)

    Luis Fang Mercado

    2012-01-01

    Full Text Available El tatuaje por amalgama se origina por el depósito en el tejido conectivo subepitelial de fragmentos de amalgama resultado de procedimientos iatrogénicos por parte del operador. La profundidad a la que se encuentren albergados los residuos de este material influye en la presentación clínica de las lesiones. Radiográficamente se pueden identificar los fragmentos mientras tengan diámetros razonables; histológicamente se pueden observar las partículas de amalgama como gránulos oscuros, sólidos e irregulares dispuesto entre los haces de colágeno y vasos sanguíneos. Este artículo refiere el caso clínico de un paciente que presentó pigmentación por amalgama en mucosa vestibular, originada por una porción de amalgama usada como material obturador en una apicectomía del 11 realizada con anterioridad. Teniendo en cuenta las consideraciones clínicas y radiográficas se optó por realizar una segunda apicectomía con obturación retrógrada con MTA del 11. Durante el procedimiento quirúrgico se cureteó y adelgazó la cara interna del colgajo mucoperióstico para tratar de disminuir el grado de pigmentación.

  13. Caracterización de la capa formada por nitruración gaseosa del titanio a alta temperatura

    Directory of Open Access Journals (Sweden)

    Rodríguez, D.

    2000-06-01

    Full Text Available Titanium and Ti6Al4V alloy are biocompatible materials. However, their lack of good wear resistance limits their use for some biomedical applications. Surface hardening by nitriding has been studied in order to improve wear resistance of titanium. The results showed an increase of surface hardness, over 300% when compared to non-treated samples. X-ray diffraction data of the surface of the treated samples showed the existence of titanium nitrides, mainly ε-nitrides (Ti2N, although δ−nitrides (TiN were also detected. Selected area diffraction pattern in transmission electron microscope (TEM of nearsurface regions detected the existence of ε-nitrides. TEM observations of regions at depths over 5 μm did not registered any nitride. Therefore, the results suggest that the studied treatment could be used to reduce wear in biomedical applications with light and medium loads.

    El titanio y la aleación Ti6Al4V son materiales que presentan una excelente biocompatibilidad, si bien la falta de una buena resistencia al desgaste dificulta su uso en diversas aplicaciones biomédicas. Se ha estudiado el endurecimiento superficial del titanio por medio de la nitruración gaseosa con el fin de mejorar su resistencia al desgaste. Los resultados obtenidos muestran un gran incremento en la dureza superficial, de un 300% con relación a las muestras no tratadas. Los datos de las difracciones de rayos X de la superficie de las muestras tratadas muestran la existencia de nitruros de titanio, principalmente nitruros-ε (Ti2N, si bien también fueron detectados nitruros-δ (TiN. La difracción de electrones realizada en microscopio electrónico de transmisión (TEM de regiones subsuperficiales de las muestras tratadas también detectaron la existencia de nitruros-ε. Observaciones con TEM de zonas a distancias de la superficie superiores a 5 μm no revelaron la existencia de nitruros. Por tanto, los resultados obtenidos indican que el tratamiento de

  14. [A new method of processing quantitative PCR data].

    Science.gov (United States)

    Ke, Bing-Shen; Li, Guang-Yun; Chen, Shi-Min; Huang, Xiang-Yan; Chen, Ying-Jian; Xu, Jun

    2003-05-01

    Today standard PCR can't satisfy the need of biotechnique development and clinical research any more. After numerous dynamic research, PE company found there is a linear relation between initial template number and cycling time when the accumulating fluorescent product is detectable.Therefore,they developed a quantitative PCR technique to be used in PE7700 and PE5700. But the error of this technique is too great to satisfy the need of biotechnique development and clinical research. A better quantitative PCR technique is needed. The mathematical model submitted here is combined with the achievement of relative science,and based on the PCR principle and careful analysis of molecular relationship of main members in PCR reaction system. This model describes the function relation between product quantity or fluorescence intensity and initial template number and other reaction conditions, and can reflect the accumulating rule of PCR product molecule accurately. Accurate quantitative PCR analysis can be made use this function relation. Accumulated PCR product quantity can be obtained from initial template number. Using this model to do quantitative PCR analysis,result error is only related to the accuracy of fluorescence intensity or the instrument used. For an example, when the fluorescence intensity is accurate to 6 digits and the template size is between 100 to 1,000,000, the quantitative result accuracy will be more than 99%. The difference of result error is distinct using same condition,same instrument but different analysis method. Moreover,if the PCR quantitative analysis system is used to process data, it will get result 80 times of accuracy than using CT method.

  15. Comparison of droplet digital PCR and seminested real-time PCR for quantification of cell-associated HIV-1 RNA

    NARCIS (Netherlands)

    Kiselinova, Maja; Pasternak, Alexander O.; de Spiegelaere, Ward; Vogelaers, Dirk; Berkhout, Ben; Vandekerckhove, Linos

    2014-01-01

    Cell-associated (CA) HIV-1 RNA is considered a potential marker for assessment of viral reservoir dynamics and antiretroviral therapy (ART) response in HIV-infected patients. Recent studies employed sensitive seminested real-time quantitative (q)PCR to quantify CA HIV-1 RNA. Digital PCR has been

  16. Transient neonatal diabetes mellitus with macroglossia diagnosed by methylation specific PCR (MS-PCR

    Directory of Open Access Journals (Sweden)

    Hye Young Jin

    2010-03-01

    Full Text Available Transient neonatal diabetes mellitus (TNDM has been associated with paternal uniparental isodisomy of chromosome 6, paternally inherited duplication of 6q24, or a methylation defect at a CpG island of the ZAC or HYMAI gene. We experienced a case of TNDM in which the patient presented with hyperglycemia, macroglossia, and intrauterine growth retardation, caused by a paternally derived HYMAI. An 18-day-old female infant was admitted to the hospital because of macroglossia and recurrent hyperglycemia. In addition to the macroglossia, she also presented with large fontanelles, micrognathia, and prominent eyes. Serum glucose levels were 200&#8211;300 mg/dL and they improved spontaneously 2 days after admission. To identify the presence of a maternal methylated allele, bisulfite-treated genomic DNA from peripheral blood was prepared and digested with BssHII after polymerase chain reaction (PCR amplification with methylation-specific HYMAI primers. PCR and restriction fragment length polymorphism analysis showed that the patient had only the paternal origin of the HYMA1 gene. TNDM is associated with a methylation defect in chromosome 6, suggesting that an imprinted gene on chromosome 6 is responsible for this phenotype.

  17. Evolution of NADPH-cytochrome P450 oxidoreductases (POR) in Apiales - POR 1 is missing

    DEFF Research Database (Denmark)

    Andersen, Trine Bundgaard; Hansen, Niels Bjørn; Laursen, Tomas

    2016-01-01

    The NADPH-dependent cytochrome P450 oxidoreductase (POR) is the obligate electron donor to eukaryotic microsomal cytochromes P450 enzymes. The number of PORs within plant species is limited to one to four isoforms, with the most common being two PORs per plant. These enzymes provide electrons to ...... (available from the SRA at NCBI). All three genes were shown to be functional upon reconstitution into nanodiscs, confirming that none of the isoforms are pseudogenes....

  18. RT-PCR Protocols - Methods in Molecular Biology

    Directory of Open Access Journals (Sweden)

    Manuela Monti

    2011-03-01

    Full Text Available “The first record I have of it, is when I made a computer file which I usually did whenever I had an idea, that would have been on the Monday when I got back, and I called it Chain Reaction.POL, meaning polymerase. That was the identifier for it and later I called the thing the Polymerase Chain Reaction, which a lot of people thought was a dumb name for it, but it stuck, and it became PCR”. With these words the Nobel prize winner, Kary Mullis, explains how he named the PCR: one of the most important techniques ever invented and currently used in molecular biology. This book “RT-PCR Protocols” covers a wide range of aspects important for the setting of a PCR experiment for both beginners and advanced users. In my opinion the book is very well structured in three different sections. The first one describes the different technologies now available, like competitive RT-PCR, nested RT-PCR or RT-PCR for cloning. An important part regards the usage of PCR in single cell mouse embryos, stressing how important...........

  19. Shape based kinetic outlier detection in real-time PCR

    Directory of Open Access Journals (Sweden)

    D'Atri Mario

    2010-04-01

    Full Text Available Abstract Background Real-time PCR has recently become the technique of choice for absolute and relative nucleic acid quantification. The gold standard quantification method in real-time PCR assumes that the compared samples have similar PCR efficiency. However, many factors present in biological samples affect PCR kinetic, confounding quantification analysis. In this work we propose a new strategy to detect outlier samples, called SOD. Results Richards function was fitted on fluorescence readings to parameterize the amplification curves. There was not a significant correlation between calculated amplification parameters (plateau, slope and y-coordinate of the inflection point and the Log of input DNA demonstrating that this approach can be used to achieve a "fingerprint" for each amplification curve. To identify the outlier runs, the calculated parameters of each unknown sample were compared to those of the standard samples. When a significant underestimation of starting DNA molecules was found, due to the presence of biological inhibitors such as tannic acid, IgG or quercitin, SOD efficiently marked these amplification profiles as outliers. SOD was subsequently compared with KOD, the current approach based on PCR efficiency estimation. The data obtained showed that SOD was more sensitive than KOD, whereas SOD and KOD were equally specific. Conclusion Our results demonstrated, for the first time, that outlier detection can be based on amplification shape instead of PCR efficiency. SOD represents an improvement in real-time PCR analysis because it decreases the variance of data thus increasing the reliability of quantification.

  20. IDENTIFIKASI TIPE HLA KELAS II DENGAN TEKNIK PCR

    Directory of Open Access Journals (Sweden)

    Ervi Salwati

    2012-09-01

    Full Text Available HLA (Human Leukocyte Antigen contains a set of genes located together on the short arm of chromosome 6. These genes control immune responses, graft acceptance or rejection and tumor surveillance. These abilities have close relationship with genetic variation (occur in "many forms" or alleles that bind and present antigens to T lymphocytes. Using advanced technology and molecular biology approaches (PCR technique detection of genetic variation in the HLA region (or HLA typing has been performed based on DNA.. PCR is an in vitro technique to amplify the DNA sequence enzymatically. "Sequence Specific Primers" (SSP are designed for this PCR to obtain amplification of specific alleles or groups of alleles. The PCR products are visualized through agarose gel electrophoresis stained with ethidium bromide. The PCR technique requires small amount of whole blood (0.5 - 1 ml, gives rapid, accurate and complete result. This paper discuss identification of HLA class II typing using PCR-SSP technique and show the examples of the results.   Key words: HLA (Human Leukocyte Antigen class II, PCR (Polymerase Chain Reaction

  1. Broad-range PCR: past, present, or future of bacteriology?

    Science.gov (United States)

    Renvoisé, A; Brossier, F; Sougakoff, W; Jarlier, V; Aubry, A

    2013-08-01

    PCR targeting the gene encoding 16S ribosomal RNA (commonly named broad-range PCR or 16S PCR) has been used for 20 years as a polyvalent tool to study prokaryotes. Broad-range PCR was first used as a taxonomic tool, then in clinical microbiology. We will describe the use of broad-range PCR in clinical microbiology. The first application was identification of bacterial strains obtained by culture but whose phenotypic or proteomic identification remained difficult or impossible. This changed bacterial taxonomy and allowed discovering many new species. The second application of broad-range PCR in clinical microbiology is the detection of bacterial DNA from clinical samples; we will review the clinical settings in which the technique proved useful (such as endocarditis) and those in which it did not (such as characterization of bacteria in ascites, in cirrhotic patients). This technique allowed identifying the etiological agents for several diseases, such as Whipple disease. This review is a synthesis of data concerning the applications, assets, and drawbacks of broad-range PCR in clinical microbiology. Copyright © 2013 Elsevier Masson SAS. All rights reserved.

  2. Ureaplasma parvum prosthetic joint infection detected by PCR.

    Science.gov (United States)

    Farrell, John J; Larson, Joshua A; Akeson, Jeffrey W; Lowery, Kristin S; Rounds, Megan A; Sampath, Rangarajan; Bonomo, Robert A; Patel, Robin

    2014-06-01

    We describe the first reported case of Ureaplasma parvum prosthetic joint infection (PJI) detected by PCR. Ureaplasma species do not possess a cell wall and are usually associated with colonization and infection of mucosal surfaces (not prosthetic material). U. parvum is a relatively new species name for certain serovars of Ureaplasma urealyticum, and PCR is useful for species determination. Our patient presented with late infection of his right total knee arthroplasty. Intraoperative fluid and tissue cultures and pre- and postoperative synovial fluid cultures were all negative. To discern the pathogen, we employed PCR coupled with electrospray ionization mass spectrometry (PCR/ESI-MS). Our patient's failure to respond to empirical antimicrobial treatment and our previous experience with PCR/ESI-MS in culture-negative cases of infection prompted us to use this approach over other diagnostic modalities. PCR/ESI-MS detected U. parvum in all samples. U. parvum-specific PCR testing was performed on all synovial fluid samples to confirm the U. parvum detection. Copyright © 2014, American Society for Microbiology. All Rights Reserved.

  3. DNA Differential Diagnosis of Taeniasis and Cysticercosis by Multiplex PCR

    Science.gov (United States)

    Yamasaki, Hiroshi; Allan, James C.; Sato, Marcello Otake; Nakao, Minoru; Sako, Yasuhito; Nakaya, Kazuhiro; Qiu, Dongchuan; Mamuti, Wulamu; Craig, Philip S.; Ito, Akira

    2004-01-01

    Multiplex PCR was established for differential diagnosis of taeniasis and cysticercosis, including their causative agents. For identification of the parasites, multiplex PCR with cytochrome c oxidase subunit 1 gene yielded evident differential products unique for Taenia saginata and Taenia asiatica and for American/African and Asian genotypes of Taenia solium with molecular sizes of 827, 269, 720, and 984 bp, respectively. In the PCR-based detection of tapeworm carriers using fecal samples, the diagnostic markers were detected from 7 of 14 and 4 of 9 T. solium carriers from Guatemala and Indonesia, respectively. Test sensitivity may have been reduced by the length of time (up to 12 years) that samples were stored and/or small sample volumes (ca. 30 to 50 mg). However, the diagnostic markers were detected by nested PCR in five worm carriers from Guatemalan cases that were found to be negative by multiplex PCR. It was noteworthy that a 720 bp-diagnostic marker was detected from a T. solium carrier who was egg-free, implying that it is possible to detect worm carriers and treat before mature gravid proglottids are discharged. In contrast to T. solium carriers, 827-bp markers were detected by multiplex PCR in all T. saginata carriers. The application of the multiplex PCR would be useful not only for surveillance of taeniasis and cysticercosis control but also for the molecular epidemiological survey of these cestode infections. PMID:14766815

  4. A multiplex PCR for detection of six viruses in ducks.

    Science.gov (United States)

    Wang, Yongjuan; Zhu, Shanyuan; Hong, Weiming; Wang, Anping; Zuo, Weiyong

    2017-10-01

    In this study, six pairs of specific primers that can amplify DNA fragments of different sizes were designed and synthesized according to viral protein gene sequences published in GenBank. Then, a multiplex PCR method was established for rapid detection of duck hepatitis virus 1, duck plague virus, duck Tembusu virus, muscovy duck parvovirus, muscovy duck reovirus, and duck H9N2 avian influenza virus, and achieve simple and rapid detection of viral diseases in ducks. Single PCR was used to confirm primer specificity, and PCR conditions were optimized to construct a multiplex PCR system. Specificity and sensitivity assays were also developed. The multiplex PCR was used to detect duck embryos infected with mixed viruses and those with clinically suspected diseases to verify the feasibility of the multiplex PCR. Results show that the primers can specifically amplify target fragments, without any cross-amplification with other viruses. The multiplex PCR system can amplify six DNA fragments from the pooled viral genomes and specifically detect nucleic acids of the six duck susceptible viruses when the template amount is 10 2 copies/μl. In addition, the system can be used to detect viral nucleic acids in duck embryos infected with the six common viruses. The detection results for clinical samples are consistent with those detected by single PCR. Therefore, the established multiplex PCR method can perform specific, sensitive, and high-throughput detection of six duck-infecting viruses and can be applied to clinical identification and diagnosis of viral infection in ducks. Copyright © 2017. Published by Elsevier B.V.

  5. PCR IN TRAUMATOLOGY AND ORTHOPAEDICS: METHOD DESCRIPTION AND APPLICABILITY

    Directory of Open Access Journals (Sweden)

    E. M. Polyakova

    2014-01-01

    Full Text Available Review brief presents description of polymerase chain reaction method (PCR and its most common variants. Three PCR-based lines of research, carried out in the traumatology and orthopaedics, include identifying a causative agents of the implant-associated infection after orthopaedic surgery; detection of antibiotic resistance genes and biofilm forming genes. It was shown that PCR can be used as additional method for detection of genetic disorders, significant for traumatology and orthopaedics, and for investigation of cartilage and bone regeneration.

  6. PCR amplification on microarrays of gel immobilized oligonucleotides

    Science.gov (United States)

    Strizhkov, Boris; Tillib, Sergei; Mikhailovich, Vladimir; Mirzabekov, Andrei

    2003-11-04

    The invention relates two general methods for performing PCR amplification, combined with the detection and analysis of the PCR products on a microchip. In the first method, the amplification occurs both outside and within a plurality of gel pads on a microchip, with at least one oligonucleotide primer immobilized in a gel pad. In the second method, PCR amplification also takes place within gel pads on a microchip, but the pads are surrounded by a hydrophobic liquid such as that which separates the individual gel pads into environments which resemble micro-miniaturized test tubes.

  7. Índices de prevalencia del ácaro Varroa destructor (Acari: Varroidae en cuadros de cría nuevos o previamente utilizados por Apis mellifera (Hymenoptera: Apidae

    Directory of Open Access Journals (Sweden)

    Jorge, A. MARCANGELI

    2007-01-01

    Full Text Available El objetivo de esta investigación fue comparar los niveles de infestación de Varroa destructor (Anderson & Trueman en panales de cría nuevos y viejos, en colonias de la abeja criolla (híbrido de Apis mellifera mellifera (Linnaeus y Apis mellifera ligustica Spinola. El trabajo se llevó a cabo en un apiario ubicado en Coronel Vidal, provincia de Buenos Aires, durante la primavera del año 2005. Se trabajó sobre 20 colmenas tipo Langstroth, de un híbrido de Apis mellifera (Linnaeus infestadas naturalmente por el ácaro Varroa destructor, y seleccionadas al azar. En cada una de ellas se escogió un panal de 2 años (viejo que se colocó en el centro del nido de cría, junto con un panal recientemente labrado por las abejas (nuevo. Luego de que ambos cuadros fueran operculados, se los extrajo y se llevaron al laboratorio para su posterior análisis. Cada una de las celdas de cría se desoperculó e inspeccionó en busca de ácaros, registrándose el número de hembras de ácaros que habían ingresado para su reproducción, se calculó el nivel de infestación como el cociente entre el número de celdas infestadas por ácaros y el número total de celdas inspeccionadas. Los resultados mostraron que los panales viejos presentaron niveles de infestación significativamente superiores a los registrados en panales nuevos (13,52% ± 3,35 y 6,18% ± 2,12 respectivamente; t = 10,62; p = 1,9 E-9; g. l.= 19. El mismo patrón fue observado en el número promedio de ácaros por panal (443,3 ± 70,54 y 217,85 ± 51,76 para panales viejos y nuevos respectivamente; t = 23,87; p = 1,24 E-15; g. l.= 19. Los ácaros presentan una marcada preferencia por los panales viejos. Esta selección estaría guiada por olores propios de las celdas, que actuarían como atrayentes. Además, posiblemente enmascaran su presencia de esta manera y evitan así ser detectados y eliminados por las abejas nodrizas mediante los comportamientos higiénicos.

  8. Intoxicação experimental por monensina em eqüinos

    Directory of Open Access Journals (Sweden)

    Bezerra Jr Pedro Soares

    2000-01-01

    Full Text Available Sete eqüinos foram tratados experimentalmente com monensina sódica. Dois desses animais receberem 3-4 kg/eqüino/dia de uma ração comercial sabidamente implicada em surtos naturais da intoxicação por monensina em eqüinos e que continha 180 ppm±20 da droga. Um eqüino recebeu uma única dose de 5 mg/kg e um outro recebeu 4 doses diárias de 1 mg/kg de monensina sódica originária de um premix. Esses quatro eqüinos morreram ou foram sacrificados in extremis, 3-8 dias após o início da administração da droga. Um quinto eqüino recebeu dose única de 5 mg/kg de monensina, ficou levemente doente e se recuperou. Dois eqüinos não desenvolveram sinais da intoxicação. Um desses eqüinos tinha recebido 40 doses diárias de 0,5 mg/kg de monensina e o outro recebeu 3 kg/dia de uma ração da mesma marca que a usada nas fazendas onde surtos de intoxicação por monensina foram detectados (mas de uma outra partida, mais tarde determinada como contendo menos de 5 ppm de monensina. O aparecimento dos sinais clínicos ocorreu de 2 a 5 dias após a administração da droga e a duração do quadro clínico variou de 24 a 76 horas. Os sinais clínicos incluíam taquicardia, arritmia, gemidos, incoordenação, sudorese, decúbito esternal, decúbito lateral, pedaleios e morte. Em cinco dos eqüinos intoxicados observaram-se marcadas elevações da atividade plasmática de creatina fosfoquinase e, em um eqüino, houve leve aumento da atividade plasmática de aspartato aminotransferase. Os principais achados de necropsia consistiram em áreas brancas ou amarelas, focais ou focalmente extensas e bilateralmente simétricas nos músculos esqueléticos. Essas lesões eram associadas a edema gelatinoso e translúcido das fáscias intermusculares. Quadríceps femoral, adutor, pectíneo, grácil, semi-membranáceo, supra-espinhal, subescapular, braquicefálico e quadríceps femoral estavam entre os músculos esqueléticos mais afetados. Os exames histol

  9. Sensitive simultaneous detection of seven sexually transmitted agents in semen by multiplex-PCR and of HPV by single PCR.

    Directory of Open Access Journals (Sweden)

    Fabrícia Gimenes

    Full Text Available Sexually transmitted diseases (STDs may impair sperm parameters and functions thereby promoting male infertility. To date limited molecular studies were conducted to evaluate the frequency and type of such infections in semen Thus, we aimed at conceiving and validating a multiplex PCR (M-PCR assay for the simultaneous detection of the following STD pathogens in semen: Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium, Trichomonas vaginalis, Herpes virus simplex (HSV -1 and -2, and Treponema pallidum; We also investigated the potential usefulness of this M-PCR assay in screening programs for semen pathogens. In addition, we aimed: to detect human Papillomavirus (HPV and genotypes by single PCR (sPCR in the same semen samples; to determine the prevalence of the seven STDs, HPV and co-infections; to assess the possibility that these infections affect semen parameters and thus fertility. The overall validation parameters of M-PCR were extremely high including agreement (99.2%, sensitivity (100.00%, specificity (99.70%, positive (96.40% and negative predictive values (100.00% and accuracy (99.80%. The prevalence of STDs was very high (55.3%. Furthermore, associations were observed between STDs and changes in semen parameters, highlighting the importance of STD detection in semen. Thus, this M-PCR assay has great potential for application in semen screening programs for pathogens in infertility and STD clinics and in sperm banks.

  10. Whole blood Nested PCR and Real-time PCR amplification of Talaromyces marneffei specific DNA for diagnosis.

    Science.gov (United States)

    Lu, Sha; Li, Xiqing; Calderone, Richard; Zhang, Jing; Ma, Jianchi; Cai, Wenying; Xi, Liyan

    2016-02-01

    Talaromyces marneffei is a dimorphic pathogenic fungus, which is a life-threatening invasive mycosis in the immunocompromised host. Prompt diagnosis of T. marneffei infection remains difficult although there has been progress in attempts to expedite the diagnosis of this infection. We previously demonstrated the value of nested polymerase chain reaction (PCR) to detect T. marneffei in paraffin embedded tissue samples with high sensitivity and specificity. In this study, this assay was used to detect the DNA of T. marneffei in whole blood samples. Real-time PCR assay was also evaluated to identify T. marneffei in the same samples. Twenty out of 30 whole blood samples (67%) collected from 23 patients were found positive by using the nested PCR assay, while 23/30 (77%) samples were found positive by using the real-time PCR assay. In order to express accurately the fungal loads, we used a normalized linearized plasmid as an internal control for real-time PCR. The assay results were correlated as the initial quantity (copies/μl) with fungal burden. These data indicate that combination of nested PCR and real-time PCR assay provides an attractive alternative for identification of T. marneffei DNA in whole blood samples of HIV-infected patients. © The Author 2015. 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.

  11. Detection of Coxiella burnetii in ticks by PCR and by PCR - Restriction Fragment Length Polymorphism (RFLP)

    International Nuclear Information System (INIS)

    2010-01-01

    Coxiella burnetii, as an obligata intracellular bacterium, is the etiologic agent of Q-fever. It is widely distributed in nature and is responsible for infection in various animals (cattle, sheep, goat) and humans. C. burnetii has been isolated from milk, ticks and human patients with acute and chronic Q fever. Ticks are the principal vectors and reservoirs of C. burnetii. Since over 40 species of ticks have been found to be infected with C. burnetii, ticks can serve as indicators of infection in nature. In this study, total of 2472 ticks (1446 female, 1021 male and 5 nymphs) were collected from 38 provinces of Turkey. The ticks were gathered into groups of 1 to 7 ticks as to the provinces, species and gender for DNA extraction. Following DNA extraction, the groups were examined for the presence of C. burtii by using the CB1and CB2. The ticks collected from the province of Denizli (56 in total) were gathered into 13 groups according to the species and gender. From these groups, 6 were positive for C. burnetii. The ticks collected from Ankara province, total of 160 ticks, were grouped into 53 as to their species and gender, only one group was found to be positive for C. burnetii. The specificities of PCR products were evaluated by restriction analysis. The positive PCR products were digested with the enzyme Taq1 and for bands in order of 118, 57, 43 and 39 bp's were appeared such as seen in the positive control DNA (C. burnetii Nine Mile RSA493)

  12. Análisis de un sistema de selección de plantas transgénicas basado en la técnica de PCR

    Directory of Open Access Journals (Sweden)

    Yomara Ivonne Rozo Peña

    2004-07-01

    propuesta viable en cuanto a implementación práctica, versatilidad y confiabilidad de la planta transgénica. El sistema de selección por PCR contribuye a la inserción competitiva de los cultivos transgénicos dentro del marco socio-económico con respecto a las variedades modificadas genéticamente producidas en la actualidad.

  13. Acuerdo por la discreción

    Directory of Open Access Journals (Sweden)

    Yeny Serrano

    2006-01-01

    Full Text Available En este artículo se propone un análisis del texto del Acuerdo por la discreción firmado en 1999 por 32 directores de medios de comunicación para “elevar el nivel de calidad y responsabilidad en el cubrimiento y difusión de hechos violentos”. Se analizan los factores que impiden que este Acuerdo produzca cambios efectivos en la práctica profesional informativa y se presenta un modelo (Lemieux, 2000 que tiene en cuenta las variables que influyen en la producción del discurso informativo mediático.

  14. ANTIOXIDANTES: MICRONUTRIENTES EN LUCHA POR LA SALUD

    OpenAIRE

    Zamora S, Juan Diego

    2007-01-01

    Los antioxidantes son sustancias químicas que se caracterizan por impedir o retrasar la oxidación de diversas sustancias principalmente de los ácidos grasos cuyas reacciones se producen tanto en los alimentos como en el organismo humano, en el cual puede provocar alteraciones fisiológicas importantes desencadenantes de diversas enfermedades. Otra de las funciones de los antioxidantes es facilitar el uso fisiológico del oxígeno por parte de las mitocondrias celulares, ayudando a reducir los ef...

  15. Alarma activada por control remoto selectivo.

    OpenAIRE

    Eguíluz Morán, Luis Ignacio; Lara Santillán, Pedro María; Mañana Canteli, Mario

    1995-01-01

    La finalidad de esta invención consiste en disponer de una alarma que bien evite el robo de objetos ligeros y portátiles por el procedimiento que se conoce como "del tirón" --es decir, arrebatar algo y salir corriendo--, bien permita identificar un equipaje entre otros. La originalidad del dispositivo reside en la activación/desactivación del dispositivo de sonido por control remoto, así como la posibilidad de seleccionar el sonido de salida dependiendo de su finalidad. Se reivindica como de ...

  16. PANORAMA LATINOAMERICANO DEL PAGO POR SERVICIOS AMBIENTALES

    Directory of Open Access Journals (Sweden)

    González T. Ángela

    2008-07-01

    Full Text Available Este documento busca proveer al lector de algunos elementos para el análisis y reflexión en torno al pago por servicios ambientales. Para ello, en primera instancia, aborda algunos conceptos básicos relacionados con economía ambiental, seguido de temas como la valoración económica de servicios ambientales y la implementación de mecanismos de pago por algunos de ellos. Lo anterior esta enriquecido con experiencias o estudios de caso a nivel latinoamericano y colombiano.

  17. DNA extraction method for PCR in mycorrhizal fungi.

    Science.gov (United States)

    Manian, S; Sreenivasaprasad, S; Mills, P R

    2001-10-01

    To develop a simple and rapid DNA extraction protocol for PCR in mycorrhizal fungi. The protocol combines the application of rapid freezing and boiling cycles and passage of the extracts through DNA purification columns. PCR amplifiable DNA was obtained from a number of endo- and ecto-mycorrhizal fungi using minute quantities of spores and mycelium, respectively. DNA extracted following the method, was used to successfully amplify regions of interest from high as well as low copy number genes. The amplicons were suitable for further downstream applications such as sequencing and PCR-RFLPs. The protocol described is simple, short and facilitates rapid isolation of PCR amplifiable genomic DNA from a large number of fungal isolates in a single day. The method requires only minute quantities of starting material and is suitable for mycorrhizal fungi as well as a range of other fungi.

  18. Some factors determining the PCr recovery overshoot in skeletal muscle.

    Science.gov (United States)

    Korzeniewski, Bernard; Zoladz, Jerzy A

    2005-07-01

    It has been proposed recently that the phosphocreatine (PCr) overshoot (increase above the resting level) during muscle recovery after exercise is caused by a slow decay during this recovery of the direct activation of oxidative phosphorylation taking place during muscle work. In the present article the factors determining the appearance and size of the PCr overshoot are studied using the computer model of oxidative phosphorylation in intact skeletal muscle developed previously. It is demonstrated that the appearance and duration of this overshoot is positively correlated with the value of the characteristic decay time of the direct activation of oxidative phosphorylation. It is also shown that the size of PCr overshoot is increased by low resting PCr/Cr ratio (what is confirmed by our unpublished experimental data), by high intensity of the direct activation of oxidative phosphorylation, by high muscle work intensity and by low rate of the return of cytosolic pH to the resting value during muscle recovery.

  19. polymerase chain reaction (pcr) provides a superior tool

    African Journals Online (AJOL)

    boaz

    Keywords: Streptococcus pneumoniae, meningitis, rt-PCR, standard bacteriological methods ... qualification de techniciens et les matériels pour son application constituent des ... Streptococcus pneumonia (pneumococcus) is a common.

  20. Multiplex polymerase chain reaction (PCR) and fluorescence-based ...

    African Journals Online (AJOL)

    reading 7

    2011-12-28

    Dec 28, 2011 ... mitochondrial DNA and cytochrome b as an internal PCR control. The amplified species- ... more labour-saving than using each pair of species- specific primers separately for .... obtained from the NCBI nucleotide data bank.

  1. Comparison of PCR-ELISA and LightCycler real-time PCR assays for detecting Salmonella spp. in milk and meat samples

    DEFF Research Database (Denmark)

    Perelle, Sylvie; Dilasser, Françoise; Malorny, Burkhard

    2004-01-01

    , minced beef and raw milk, and 92 naturally-contaminated milk and meat samples. When using either PCR-ELISA or LC-PCR assays, only Salmonella strains were detected. PCR-ELISA and LC-PCR assays gave with pure Salmonella cultures the same detection limit level of 10(3) CFU/ml, which corresponds respectively...

  2. Development of RT-PCR and Nested PCR for Detecting Four Quarantine Plant Viruses Belonging to Nepovirus

    Directory of Open Access Journals (Sweden)

    Siwon Lee

    2013-09-01

    Full Text Available For quarantine purpose, we developed the RT- and nested PCR module of Tomato black ring virus (TBRV, Arabis mosaic virus (ArMV, Cherry leafroll virus (CLRV and Grapevine fanleaf virus (GFLV. The PCR modules, developed in this study make diagnosis more convenient and speedy because of same PCR condition. And also, the methods are more accurate because it can check whether the result is contamination or not using the mutation-positive control. We discard or return the 27 cases of Nepovirus infection seed by employing the module past 3 years. This study provides a rapid and useful method for detection of four quarantine plant viruses.

  3. Estandarización de la PCR para el diagnóstico del virus de la Hepatitis B en el Perú

    Directory of Open Access Journals (Sweden)

    G Hijar

    1998-01-01

    Full Text Available La detección del ADN del virus de la Hepatitis B, (VHB es el marcador más sensible para determinar la replicación activa e infectividad del virus circulante. Por esta razón la Reacción en Cadena de la Polimerasa (PCR fue aplicada satisfactoriamente usando primers específicos para gen del antígeno de superficie (HBsAg. Un fragmento de 260 bp fue amplificado in vitro a partir de 200 µl. de sueros de pacientes aplicando PCR. La tecnología de PCR está siendo aplicada en el diagnóstico de pacientes infectados con el virus de la Hepatitis B pertenecientes a diferentes zonas geográficas.

  4. Culture independent PCR: an alternative enzyme discovery strategy

    DEFF Research Database (Denmark)

    Jacobsen, Jonas; Lydolph, Magnus; Lange, Lene

    2005-01-01

    Degenerate primers were designed for use in a culture-independent PCR screening of DNA from composite fungal communities, inhabiting residues of corn stovers and leaves. According to similarity searches and alignments amplified clone sequences affiliated with glycosyl hydrolase family 7 and glyco...... the value of culture-independent PCR in microbial diversity studies and could add to development of a new enzyme screening technology....

  5. MPprimer: a program for reliable multiplex PCR primer design

    Directory of Open Access Journals (Sweden)

    Wang Xiaolei

    2010-03-01

    Full Text Available Abstract Background Multiplex PCR, defined as the simultaneous amplification of multiple regions of a DNA template or multiple DNA templates using more than one primer set (comprising a forward primer and a reverse primer in one tube, has been widely used in diagnostic applications of clinical and environmental microbiology studies. However, primer design for multiplex PCR is still a challenging problem and several factors need to be considered. These problems include mis-priming due to nonspecific binding to non-target DNA templates, primer dimerization, and the inability to separate and purify DNA amplicons with similar electrophoretic mobility. Results A program named MPprimer was developed to help users for reliable multiplex PCR primer design. It employs the widely used primer design program Primer3 and the primer specificity evaluation program MFEprimer to design and evaluate the candidate primers based on genomic or transcript DNA database, followed by careful examination to avoid primer dimerization. The graph-expanding algorithm derived from the greedy algorithm was used to determine the optimal primer set combinations (PSCs for multiplex PCR assay. In addition, MPprimer provides a virtual electrophotogram to help users choose the best PSC. The experimental validation from 2× to 5× plex PCR demonstrates the reliability of MPprimer. As another example, MPprimer is able to design the multiplex PCR primers for DMD (dystrophin gene which caused Duchenne Muscular Dystrophy, which has 79 exons, for 20×, 20×, 20×, 14×, and 5× plex PCR reactions in five tubes to detect underlying exon deletions. Conclusions MPprimer is a valuable tool for designing specific, non-dimerizing primer set combinations with constrained amplicons size for multiplex PCR assays.

  6. Template preparation for rapid PCR in Colletotrichum lindemuthianum

    Directory of Open Access Journals (Sweden)

    Roca M. Gabriela

    2003-01-01

    Full Text Available Isolation of DNA for PCR is time-consuming and involves many reagents. The aim of this work was to optimise a rapid and easy PCR methodology without previous DNA isolation. Different strains of the phytopathogenic fungus Colletotrichum lindemuthianum were used. Protoplasts were generated using lytic enzymes under high incubation temperatures using different methodologies to obtain the template. A rapid (10 minute methodology was successful for smaller amplicons (<750 bp.

  7. DNA microarray-based PCR ribotyping of Clostridium difficile.

    Science.gov (United States)

    Schneeberg, Alexander; Ehricht, Ralf; Slickers, Peter; Baier, Vico; Neubauer, Heinrich; Zimmermann, Stefan; Rabold, Denise; Lübke-Becker, Antina; Seyboldt, Christian

    2015-02-01

    This study presents a DNA microarray-based assay for fast and simple PCR ribotyping of Clostridium difficile strains. Hybridization probes were designed to query the modularly structured intergenic spacer region (ISR), which is also the template for conventional and PCR ribotyping with subsequent capillary gel electrophoresis (seq-PCR) ribotyping. The probes were derived from sequences available in GenBank as well as from theoretical ISR module combinations. A database of reference hybridization patterns was set up from a collection of 142 well-characterized C. difficile isolates representing 48 seq-PCR ribotypes. The reference hybridization patterns calculated by the arithmetic mean were compared using a similarity matrix analysis. The 48 investigated seq-PCR ribotypes revealed 27 array profiles that were clearly distinguishable. The most frequent human-pathogenic ribotypes 001, 014/020, 027, and 078/126 were discriminated by the microarray. C. difficile strains related to 078/126 (033, 045/FLI01, 078, 126, 126/FLI01, 413, 413/FLI01, 598, 620, 652, and 660) and 014/020 (014, 020, and 449) showed similar hybridization patterns, confirming their genetic relatedness, which was previously reported. A panel of 50 C. difficile field isolates was tested by seq-PCR ribotyping and the DNA microarray-based assay in parallel. Taking into account that the current version of the microarray does not discriminate some closely related seq-PCR ribotypes, all isolates were typed correctly. Moreover, seq-PCR ribotypes without reference profiles available in the database (ribotype 009 and 5 new types) were correctly recognized as new ribotypes, confirming the performance and expansion potential of the microarray. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

  8. Diagnosis of aerobic vaginitis by quantitative real-time PCR

    OpenAIRE

    Rumyantseva, T. A.; Bellen, G.; Savochkina, Y. A.; Guschin, A. E.; Donders, G.G.G.

    2016-01-01

    Abstract: Purpose To evaluate a real-time PCR-based technique to quantify bacteria associated with aerobic vaginitis (AV) as a potential test. Methods Vaginal samples from 100 women were tested by wet-mount microscopy, gram stain and quantitative real-time PCR targeting Enterobacteriacea, Staphylococcus spp., Streptococcus spp., Enterococcus spp., Escherichia coli, Streptococcus agalactiae, S. aureus; Lactobacillus spp. AV diagnosis obtained by wet-mount microscopy was used as reference. Resu...

  9. [Optimized application of nested PCR method for detection of malaria].

    Science.gov (United States)

    Yao-Guang, Z; Li, J; Zhen-Yu, W; Li, C

    2017-04-28

    Objective To optimize the application of the nested PCR method for the detection of malaria according to the working practice, so as to improve the efficiency of malaria detection. Methods Premixing solution of PCR, internal primers for further amplification and new designed primers that aimed at two Plasmodium ovale subspecies were employed to optimize the reaction system, reaction condition and specific primers of P . ovale on basis of routine nested PCR. Then the specificity and the sensitivity of the optimized method were analyzed. The positive blood samples and examination samples of malaria were detected by the routine nested PCR and the optimized method simultaneously, and the detection results were compared and analyzed. Results The optimized method showed good specificity, and its sensitivity could reach the pg to fg level. The two methods were used to detect the same positive malarial blood samples simultaneously, the results indicated that the PCR products of the two methods had no significant difference, but the non-specific amplification reduced obviously and the detection rates of P . ovale subspecies improved, as well as the total specificity also increased through the use of the optimized method. The actual detection results of 111 cases of malarial blood samples showed that the sensitivity and specificity of the routine nested PCR were 94.57% and 86.96%, respectively, and those of the optimized method were both 93.48%, and there was no statistically significant difference between the two methods in the sensitivity ( P > 0.05), but there was a statistically significant difference between the two methods in the specificity ( P PCR can improve the specificity without reducing the sensitivity on the basis of the routine nested PCR, it also can save the cost and increase the efficiency of malaria detection as less experiment links.

  10. Hepatite crônica por vírus C: Parte 1. Considerações gerais Hepatitis C virus: Part 1. General considerations

    Directory of Open Access Journals (Sweden)

    Vinício Paride CONTE

    2000-07-01

    necessariamente ausência de vírus da hepatite C no fígado. A associação de autoimunidade à hepatite C é questionável. Os marcadores imunes são freqüentemente detectados em baixos níveis. A modulação da resposta imune ao envelope protéico E2, após injeção de plasmídios DNA, tem sido usada para induzir respostas imunes específicas ao vírus da hepatite C. O espectro de tais respostas poderia ser ampliada mediante combinação de plasmídios, vias de administração e outras formas de imunógenos codificados (vacinas a base de peptídios. Tais estratégias podem vir a ser importantes, em breve, no combate aos altamente mutantes vírus da hepatite C. O papel patogênico dos novos vírus G e TT das hepatites está em estudos, porém é de consenso que suas associações com doença hepática ativa é fortuita.Hepatitis C virus was identified in 1989 as the main causative agent of non-A, non-B and was followed by the recognition of a high prevalence of hepatitis C virus infection after transfusion of infected blood or blood products and in association with intravenous drug abuse. The availability of sensitive and reliable techniques to screen blood for hepatitis C virus has reduced the incidence of post-transfusion hepatitis. True healthy carriers of hepatitis C virus did not exist. Aproximately 95% of hepatitis C virus infected individuals can be identified by third generation anti- hepatitis C virus testing. Retrospective studies of iatrogenic hepatitis C virus infection are the main source of the natural history of the disease. The distribution of different hepatitis C virus genotypes varies according to the grographic region. In South America, Europe, The United States and Japan hepatitis C virus genotypes 1, 2 and 3 account for the majority of the infections, being (subtype 1b the most prevalent. Epidemiological parameters (age, risk factors and duration of infection may be associated with hepatitis C virus genotypes (intravenous drug abuse with types 1-a and 3

  11. The use of acetonitrile as the sole nitrogen and carbon source by Geotrichum sp. JR1 Uso de acetonitrila como única fonte de carbono e nitrogênio por Geotrichum sp. JR1

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    Rachel Passos Rezende

    2004-06-01

    Full Text Available A yeast strain identified as Geotrichum sp. JR1 was able to use acetonitrile as the sole carbon and nitrogen source. The strain grew in 0.5 to 2M acetonitrile. Ammonia generation as enzymatic product during the strain growth indicates the presence of an acetonitrile degrading enzyme. Acetic acid and acetamide were detected during assays with the resting cells cultivated in acetonitrile, indicating the presence of nitrile and amide degrading enzymes. This paper is the first to describe the use of acetonitrile as the sole carbon and nitrogen source by a yeast.Uma linhagem de levedura identificada como Geotrichum sp. JR1 foi capaz de utilizar acetonitrila, em concentrações de 0,5 a 2M, como única fonte de carbono e de nitrogênio. A geração de amônia durante o crescimento do microrganismo indica a presença de sistema enzimático capaz de degradar acetonitrila. Durante os ensaios enzimáticos, com células cultivadas em acetonitrila, foram detectados ácido acético e acetamida como produtos indicando a presença de sistema enzimático capaz de degradar acetonitrila e acetamida. Este trabalho é o primeiro a descrever a utilização de acetonitrila como única fonte de carbono e de nitrogênio por uma levedura.

  12. PCR performance of a thermostable heterodimeric archaeal DNA polymerase

    Science.gov (United States)

    Killelea, Tom; Ralec, Céline; Bossé, Audrey; Henneke, Ghislaine

    2014-01-01

    DNA polymerases are versatile tools used in numerous important molecular biological core technologies like the ubiquitous polymerase chain reaction (PCR), cDNA cloning, genome sequencing, and nucleic acid based diagnostics. Taking into account the multiple DNA amplification techniques in use, different DNA polymerases must be optimized for each type of application. One of the current tendencies is to reengineer or to discover new DNA polymerases with increased performance and broadened substrate spectra. At present, there is a great demand for such enzymes in applications, e.g., forensics or paleogenomics. Current major limitations hinge on the inability of conventional PCR enzymes, such as Taq, to amplify degraded or low amounts of template DNA. Besides, a wide range of PCR inhibitors can also impede reactions of nucleic acid amplification. Here we looked at the PCR performances of the proof-reading D-type DNA polymerase from P. abyssi, Pab-polD. Fragments, 3 kilobases in length, were specifically PCR-amplified in its optimized reaction buffer. Pab-polD showed not only a greater resistance to high denaturation temperatures than Taq during cycling, but also a superior tolerance to the presence of potential inhibitors. Proficient proof-reading Pab-polD enzyme could also extend a primer containing up to two mismatches at the 3' primer termini. Overall, we found valuable biochemical properties in Pab-polD compared to the conventional Taq, which makes the enzyme ideally suited for cutting-edge PCR-applications. PMID:24847315

  13. PCR performance of a thermostable heterodimeric archaeal DNA polymerase

    Directory of Open Access Journals (Sweden)

    Tom eKillelea

    2014-05-01

    Full Text Available DNA polymerases are versatile tools used in numerous important molecular biological core technologies like the ubiquitous polymerase chain reaction (PCR, cDNA cloning, genome sequencing and nucleic acid based diagnostics. Taking into account the multiple DNA amplification techniques in use, different DNA polymerases must be optimized for each type of application. One of the current tendencies is to reengineer or to discover new DNA polymerases with increased performance and broadened substrate spectra. At present, there is a great demand for such enzymes in applications, e.g., forensics or paleogenomics. Current major limitations hinge on the inability of conventional PCR enzymes, such as Taq, to amplify degraded or low amounts of template DNA. Besides, a wide range of PCR inhibitors can also impede reactions of nucleic acid amplification. Here we looked at the PCR performances of the proof-reading D-type DNA polymerase from P. abyssi, Pab-polD. Fragments, 3 kilobases in length, were specifically PCR-amplified in its optimized reaction buffer. Pab-polD showed not only a greater resistance to high denaturation temperatures than Taq during cycling, but also a superior tolerance to the presence of potential inhibitors. Proficient proof-reading Pab-polD enzyme could also extend a primer containing up to two mismatches at the 3’ primer termini. Overall, we found valuable biochemical properties in Pab-polD compared to the conventional Taq, which makes the enzyme ideally suited for cutting-edge PCR-applications.

  14. Immunomagnetic nanoparticle based quantitative PCR for rapid detection of Salmonella

    International Nuclear Information System (INIS)

    Bakthavathsalam, Padmavathy; Rajendran, Vinoth Kumar; Saran, Uttara; Chatterjee, Suvro; Ali, Baquir Mohammed Jaffar

    2013-01-01

    We have developed a rapid and sensitive method for immunomagnetic separation (IMS) of Salmonella along with their real time detection via PCR. Silica-coated magnetic nanoparticles were functionalized with carboxy groups to which anti-Salmonella antibody raised against heat-inactivated whole cells of Salmonella were covalently attached. The immuno-captured target cells were detected in beverages like milk and lemon juice by multiplex PCR and real time PCR with a detection limit of 10 4 cfu.mL −1 and 10 3 cfu.mL −1 , respectively. We demonstrate that IMS can be used for selective concentration of target bacteria from beverages for subsequent use in PCR detection. PCR also enables differentiation of Salmonella typhi and Salmonella paratyphi A using a set of four specific primers. In addition, IMS—PCR can be used as a screening tool in the food and beverage industry for the detection of Salmonella within 3–4 h which compares favorably to the time of several days that is needed in case of conventional detection based on culture and biochemical methods. (author)

  15. IDENTIFIKASI DAGING BABI MENGGUNAKAN METODE PCR-RFLP GEN Cytochrome b DAN PCR PRIMER SPESIFIK GEN AMELOGENIN (Pork Identification Using PCR-RFLP of Cytochrome b Gene and Species Specific PCR of Amelogenin Gene

    Directory of Open Access Journals (Sweden)

    Yuny Erwanto

    2013-03-01

    Full Text Available A polymerase chain reaction–restriction fragment length polymorphism (PCR–RFLP and species specific PCR methods had been applied for identifying pork in mixture of meat. Pork sample in various levels (1, 3, 5 and 10% was prepared in mixture with beef, chicken and mutton. The primary CYTb1 and CYTb2 were designed in the mitochondrial cytochrome b b (cytochrome b gene and PCR successfully amplified fragments of 359 bp. To distinguish pig species existence, the amplified PCR products of mitochondrial DNA were cut by BseDI restriction enzyme. The result showed that pig mitochondrial DNA was cut into 131 and 228 bp fragments. A polymerase chain reaction (PCR method based on the nucleotide sequence variation in the amelogenin gene has been chosen for the specific identification of pork DNAs in mixture meat. The primers designed generated specific fragments of 353 and 312 bp length for pork. The specificity of the primary designed was tested on 4 animal species including pig, cattle, chicken and goat species. Analysis of experimental mixture meat demonstrated that 1% of raw pork tissues could be detected using PCR-RFLP with BseDI restriction enzyme but detection using species-specific PCR showed the cross reactivity to beef, chicken and mutton. The cytochrome b PCR-RFLP species identification assay yielded excellent results for identification of pig species. PCR-RFLP is a potentially reliable technique for detection of the existence of pork in animal food product for Halal authentication. Keywords: Pork identification, cytochrome b, amelogenin, polymerase chain reaction   ABSTRAK   Penelitian ini dilakukan untuk mengaplikasikan metode deteksi daging babi dalam campuan daging dengan sapi, kambing dan ayam melalui PCR-RFLP dan PCR dengan primer spesifik untuk babi. Level kontaminasi daging babi dibuat sebesar 1, 3, 5 dan 10% dari total daging dalam campuran. Metode PCR-RFLP menggunakan sepasang primer yaitu gen cytochrome b dari mitokondria yang

  16. Pedro Teixeira y su viaje por Mesopotamia

    Directory of Open Access Journals (Sweden)

    Fuente del Pilar, José Javier

    2005-04-01

    Full Text Available Pedro Teixeira es un integrante notable de la ilustre nómina de los viajeros portugueses que, a finales del s, XVI y principios del XVII, ensancharon para Occidente las fronteras del mundo. Su conocimiento en España se debe a la publicación en 1994 de su obra «Relaciones del Origen, Descendencia y Sucesión de los Reyes de Persia, y de Harmuz, y de un viaje hecho por el autor dende la India hasta Italia por tierra», en edición realizada por el profesor Eduardo Barajas Sala, lamentablemente fallecido en 1997. En este artículo se ofrece una reseña biográfica de Pedro Teixeira, y un análisis del viaje narrado por el autor en la última parte de sus «Relaciones »: el que desde Ormuz le conducirá, a través de Mesopotamia, hasta la costa del Mediterráneo.…

  17. Comparative validation using quantitative real-time PCR (qPCR and conventional PCR of bovine semen centrifuged in continuous density gradient

    Directory of Open Access Journals (Sweden)

    M.V. Resende

    2011-06-01

    Full Text Available The objective of the present study was to determine the sperm enrichment with X-bearing spermatozoa, after one centrifugation in a Percoll or OptiPrep continuous density gradient, using quantitative real-time polymerase chain reaction (qPCR of sperm DNA and resultant in vitro-produced bovine embryos by PCR. Frozen/thawed sperm was layered on density gradients and the tubes were centrifuged. Supernatants were gently aspirated and the sperm recovered from the bottom of the tubes. Cleavage and blastocyst rates were determined through in vitro production of embryos and PCR was performed to identify the embryos' genetic sex. A difference in blastocyst rate was found in the Percoll treatment compared to OptiPrep (P<0.05. The percentage of female embryos in the Percoll and OptiPrep groups was 62.0% and 47.1%, respectively. These results were confirmed by qPCR of spermatozoa DNA and underestimation was seen only in the Percoll group. It was possible to sexing sperm using simple approach.

  18. RT-PCR Detection of HIV in Republic of Macedonia

    Directory of Open Access Journals (Sweden)

    Golubinka Bosevska

    2008-11-01

    Full Text Available The aim of the study was to detect HIV RNA in seropositive patients using RT-PCR method and thus, to establish PCR methodology in the routine laboratory works.The total of 33 examined persons were divided in two groups: 1 13 persons seropositive for HIV; and 2 20 healthy persons - randomly selected blood donors that made the case control group. The subjects age was between 25 and 52 years (average 38,5.ELFA test for combined detection of HIV p24 antigen and anti HIV-1 + 2 IgG and ELISA test for detection of antibodies against HIV-1 and HIV-2, were performed for each examined person. RNA from the whole blood was extracted using a commercial kit based on salt precipitation. Detection of HIV RNA was performed using RT-PCR kit. Following nested PCR, the product was separated by electrophoresis in 1,5 % agarose gel. The result was scored positive if the band of 210bp was visible regardless of intensity Measures of precaution were taken during all the steps of the work and HIV infected materials were disposed of accordingly.In the group of blood donors ELFA, ELISA and RT-PCR were negative. Assuming that prevalence of HIV infection is zero, the clinical specificity of RT-PCR is 100 %. The analytical specificity of RT-PCR method was tested against Hepatitis C and B, Human Papiloma Virus, Cytomegalovirus, Herpes Simplex Virus, Rubella Virus, Mycobacterium tuberculosis, Chlamydia trachomatis. None of these templates yielded amplicon. In the group of 13 seropositive persons, 33 samples were analyzed. HIV RNA was detected in 15 samples. ELISA and ELFA test were positive in all samples. Different aliquots of the samples were tested independently and showed the same results. After different periods of storing the RNA samples at -70°C, RT-PCR reaction was identical to the one performed initially. The obtained amplicons were maintained frozen at -20°C for a week and the subsequently performed electrophoresis was identical to the previous one. The reaction is

  19. Detection of Streptococcus pneumoniae in whole blood by PCR.

    Science.gov (United States)

    Zhang, Y; Isaacman, D J; Wadowsky, R M; Rydquist-White, J; Post, J C; Ehrlich, G D

    1995-03-01

    Streptococcus pneumoniae is a major cause of bacteremia in both children and adults. Currently, the diagnosis of pneumococcal bacteremia relies on the isolation and identification of the bacteria from blood cultures. We have developed a sensitive assay for the detection of S. pneumoniae in whole blood by the PCR. A specific primer-probe set (JM201 and JM202 primers with JM204 probe) designed from the penicillin-binding protein 2B gene was demonstrated to reproducibly detect between 10 and 100 fg of input purified S. pneumoniae DNA. This assay system was shown to be inclusive for all strains of S. pneumoniae evaluated, including 15 different serotypes and a battery of penicillin-resistant and -sensitive strains. The specificity of this PCR-based assay was demonstrated by its inability to support amplification from a series of human, bacterial, and yeast genomic DNAs. A general specimen preparation method which should be suitable for the purification of DNA from any pathogens in whole blood was developed. With this protocol it was possible to detect S. pneumoniae-specific DNA from whole blood specimens inoculated with as little as 4 CFU/ml. Copurified human blood DNA, ranging from 0 to 4.5 micrograms per PCR, did not affect the sensitivity of S. pneumoniae detection by PCR. A blinded clinical trial was used to compare the PCR-based assay with standard microbiological blood culture for the detection of S. pneumoniae bacteremia in 36 specimens obtained from pediatric patients seen in the emergency room of Children's Hospital of Pittsburgh. With culture as the "gold standard," the PCR-based assay had a sensitivity of 80% (4 of 5 culture-positive specimens were PCR positive) and a specificity of 84% (26 of 31 culture-negative specimens were PCR negative). However, three patients whose specimens were PCR positive and culture negative had histories suggestive of bacteremia, including recent positive blood cultures, treatment with antibiotics, cellulitis, and multiple

  20. Species Identification of Fox-, Mink-, Dog-, and Rabbit-Derived Ingredients by Multiplex PCR and Real-Time PCR Assay.

    Science.gov (United States)

    Wu, Qingqing; Xiang, Shengnan; Wang, Wenjun; Zhao, Jinyan; Xia, Jinhua; Zhen, Yueran; Liu, Bang

    2018-05-01

    Various detection methods have been developed to date for identification of animal species. New techniques based on PCR approach have raised the hope of developing better identification methods, which can overcome the limitations of the existing methods. PCR-based methods used the mitochondrial DNA (mtDNA) as well as nuclear DNA sequences. In this study, by targeting nuclear DNA, multiplex PCR and real-time PCR methods were developed to assist with qualitative and quantitative analysis. The multiplex PCR was found to simultaneously and effectively distinguish four species (fox, dog, mink, and rabbit) ingredients by the different sizes of electrophoretic bands: 480, 317, 220, and 209 bp. Real-time fluorescent PCR's amplification profiles and standard curves showed good quantitative measurement responses and linearity, as indicated by good repeatability and coefficient of determination R 2  > 0.99. The quantitative results of quaternary DNA mixtures including mink, fox, dog, and rabbit DNA are in line with our expectations: R.D. (relative deviation) varied between 1.98 and 12.23% and R.S.D. (relative standard deviation) varied between 3.06 and 11.51%, both of which are well within the acceptance criterion of ≤ 25%. Combining the two methods is suitable for the rapid identification and accurate quantification of fox-, dog-, mink-, and rabbit-derived ingredients in the animal products.

  1. Analysis of ELA-DQB exon 2 polymorphism in Argentine Creole horses by PCR-RFLP and PCR-SSCP.

    Science.gov (United States)

    Villegas-Castagnasso, E E; Díaz, S; Giovambattista, G; Dulout, F N; Peral-García, P

    2003-08-01

    The second exon of equine leucocyte antigen (ELA)-DQB genes was amplified from genomic DNA of 32 Argentine Creole horses by PCR. Amplified DNA was analysed by PCR-restriction fragment length polymorphism (RFLP) and PCR-single-strand conformation polymorphism (SSCP). The PCR-RFLP analysis revealed two HaeIII patterns, four RsaI patterns, five MspI patterns and two HinfI patterns. EcoRI showed no variation in the analysed sample. Additional patterns that did not account for known exon 2 DNA sequences were observed, suggesting the existence of novel ELA-DQB alleles. PCR-SSCP analysis exhibited seven different band patterns, and the number of bands per animal ranged from four to nine. Both methods indicated that at least two DQB genes are present. The presence of more than two alleles in each animal showed that the primers employed in this work are not specific for a unique DQB locus. The improvement of this PCR-RFLP method should provide a simple and rapid technique for an accurate definition of ELA-DQB typing in horses.

  2. Inhibitory effect of common microfluidic materials on PCR outcome

    KAUST Repository

    Kodzius, Rimantas

    2012-02-20

    Microfluidic chips have a variety of applications in the biological sciences and medicine. In contrast with traditional experimental approaches, microfluidics entails lower sample and reagent consumption, allows faster reactions and enables efficient separation. Additionally microfluidics offers other advantages accruing from the fluids’ various distinct behaviors, such as energy dissipation, fluidic resistance, laminar flow, and surface tension. Biological molecules suspended in fluid and transported through microfluidics channels interact with the channel-wall material. This interaction is even stronger in high surface-area-to-volume ratio (SAVR) microfluidic channels. Adsorption and inhibition of biomolecules occur when these materials come in contact with biomolecular reaction components. Polymerase chain reaction (PCR) is a thermal cycling procedure for amplifying target DNA. The PCR compatibility of silicon, silicon dioxide (SiO2) and other surfaces have been studied; however the results are inconclusive. Usually for protein-surface interaction measurements, bulky and expensive equipment is used, such as Atomic Force Microscopy (AFM), Scanning or Transmission Electron Microscopy (SEM, TEM), spectrophotometric protein concentration measurement, Fourier transform infrared spectroscopy (FTIR) or X-Ray photoelectron spectroscopy (XPS). \\tThe PCR reaction components include the DNA template, primers, DNA polymerase (the main component), dNTPs, a buffer, divalent ions (MgCl2), and KCl. \\tWe designed a simple, relatively quick measurement that only requires a PCR cycler; thus it mimics actual conditions in PCR cycling. In our study, we evaluated the inhibitory affect of different materials on PCR, which is one of the most frequently used enzymatic reactions in microfluidics. PCR reaction optimization through choice of surface materials is of the upmost importance, as it enables and improves enzymatic reaction in microfluidics. Our assessment of the PCR

  3. Enfermedad neurologica por adenovirus Neurologic disease due to adenovirus infection

    Directory of Open Access Journals (Sweden)

    Cristina L. Lema

    2005-06-01

    Full Text Available El objetivo de este trabajo fue determinar la prevalencia de adenovirus (ADV en las infecciones del sistema nervioso central (SNC. Se analizaron 108 muestras de líquido cefalorraquídeo (LCR provenientes de 79 casos de encefalitis, 7 meningitis y 22 de otras patologías neurológicas, recibidas en el período 2000-2002. Cuarenta y nueve (47.35% se obtuvieron de pacientes inmunocomprometidos. La presencia de ADV se investigó mediante reacción en cadena de la polimerasa en formato anidado (Nested-PCR. La identificación del genogrupo se realizó mediante análisis filogenético de la secuencia nucleotídica parcial de la región que codifica para la proteína del hexón. Se detectó la presencia de ADV en 6 de 108 (5.5% muestras de LCR analizadas. Todos los casos positivos pertenecieron a pacientes con encefalitis que fueron 79, (6/79, 7.6%. No se observó diferencia estadísticamente significativa entre los casos de infección por ADV en pacientes inmunocomprometidos e inmunocompetentes (p>0.05. Las cepas de ADV detectadas se agruparon en los genogrupos B1 y C. En conclusión, nuestros resultados describen el rol de los ADV en las infecciones neurológicas en Argentina. La información presentada contribuye al conocimiento de su epidemiología, en particular en casos de encefalitis.The aim of this study was to assess the prevalence of adenovirusm (ADV infections in neurological disorders. A total of 108 cerebrospinal fluid (CSF samples from 79 encephalitis cases, 7 meningitis and 22 other neurological diseases analysed in our laboratory between 2000 and 2002 were studied. Forty nine (47.4% belonged to immunocompromised patients. Viral genome was detected using nested polymerase chain reaction (Nested-PCR and ADV genotypes were identified using partial gene sequence analysis of hexon gene. Adenovirus were detected in 6 of 108 (5.5% CSF samples tested. All of these were from encephalitis cases, 6/79, representing 7.6% of them. No statistically

  4. Droplet digital PCR (ddPCR) vs quantitative real-time PCR (qPCR) approach for detection and quantification of Merkel cell polyomavirus (MCPyV) DNA in formalin fixed paraffin embedded (FFPE) cutaneous biopsies.

    Science.gov (United States)

    Arvia, Rosaria; Sollai, Mauro; Pierucci, Federica; Urso, Carmelo; Massi, Daniela; Zakrzewska, Krystyna

    2017-08-01

    Merkel cell polyomavirus (MCPyV) is associated with Merkel cell carcinoma and high viral load in the skin was proposed as a risk factor for the occurrence of this tumour. MCPyV DNA was detected, with lower frequency, in different skin cancers but since the viral load was usually low, the real prevalence of viral DNA could be underestimated. To evaluate the performance of two assays (qPCR and ddPCR) for MCPyV detection and quantification in formalin fixed paraffin embedded (FFPE) tissue samples. Both assays were designed to simultaneous detection and quantification of both MCPyV as well as house-keeping DNA in clinical samples. The performance of MCPyV quantification was investigated using serial dilutions of cloned target DNA. We also evaluated the applicability of both tests for the analysis of 76 FFPE cutaneous biopsies. The two approaches resulted equivalent with regard to the reproducibility and repeatability and showed a high degree of linearity in the dynamic range tested in the present study. Moreover, qPCR was able to quantify ≥10 5 copies per reaction, while the upper limit of ddPCR was 10 4 copies. There was not significant difference between viral load measured by the two methods The detection limit of both tests was 0,15 copies per reaction, however, the number of positive samples obtained by ddPCR was higher than that obtained by qPCR (45% and 37% respectively). The ddPCR represents a better method for detection of MCPyV in FFPE biopsies, mostly these containing low copies number of viral genome. Copyright © 2017 Elsevier B.V. All rights reserved.

  5. Primeiro relato no Brasil de mastite necrótica bovina por Clostridium perfringens tipo A First report in Brazil of bovine necrotic mastitis due to Clostridium perfringens type A

    Directory of Open Access Journals (Sweden)

    Luciana Aramuni Gonçalves

    2006-08-01

    Full Text Available Relata-se o primeiro caso no Brasil de mastite bovina por Clostridium perfringens tipo A. O quadro clínico caracterizou-se por necrose da papila mamária e porção ventral do quarto afetado. O agente foi isolado em cultura pura e identificado como tipo A por PCR a partir do leite do quarto mamário afetado.This report describes a case of bovine mastitis due to Clostridium perfringens type A for first time in Brazil. The unical case showed necrosis of papilla mammary and ventral portion of the affected quarter. The microorganism was isolated in pure culture and identified as type A by PCR from milk of the affected mammary quarter.

  6. Species identification of Cannabis sativa using real-time quantitative PCR (qPCR).

    Science.gov (United States)

    Johnson, Christopher E; Premasuthan, Amritha; Satkoski Trask, Jessica; Kanthaswamy, Sree

    2013-03-01

    Most narcotics-related cases in the United States involve Cannabis sativa. Material is typically identified based on the cystolithic hairs on the leaves and with chemical tests to identify of the presence of cannabinoids. Suspect seeds are germinated into a viable plant so that morphological and chemical tests can be conducted. Seed germination, however, causes undue analytical delays. DNA analyses that involve the chloroplast and nuclear genomes have been developed for identification of C. sativa materials, but they require several nanograms of template DNA. Using the trnL 3' exon-trnF intragenic spacer regions within the C. sativa chloroplast, we have developed a real-time quantitative PCR assay that is capable of identifying picogram amounts of chloroplast DNA for species determination of suspected C. sativa material. This assay provides forensic science laboratories with a quick and reliable method to identify an unknown sample as C. sativa. © 2013 American Academy of Forensic Sciences.

  7. La infección por virus Zika: una nueva emergencia de salud pública con gran impacto mediático

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    Joan A. Caylà

    2016-11-01

    Full Text Available La infección por virus Zika (VZ está afectando intensamente a los países latinoamericanos y se ha convertido en una nueva epidemia mediática. Su posible asociación con microcefalia y síndrome de Guillain-Barré motivó que la Organización Mundial de la Salud (OMS declarase el 1 de febrero de 2016 que esta epidemia constituye una emergencia de salud pública de importancia internacional. Los datos epidemiológicos muestran una incidencia creciente en países como Brasil y Colombia, y que la epidemia sigue expandiéndose por muchos otros países. Desde enero de 2007 hasta el 27 de abril de 2016, la OMS ha detectado transmisión autóctona en 55 países (en 42 de ellos ha sido el primer brote de Zika, y 1198 microcefalias y otros trastornos neurológicos en Brasil. Así mismo, durante 2015 y 2016, 13 países detectaron un incremento de los casos de síndrome de Guillain-Barré y de confirmación de VZ asociado a este. En relación a las microcefalias y otras graves alteraciones cerebrales en recién nacidos de madres afectadas por VZ, las investigaciones ya evidencian una relación causal. Clínicamente muchos casos son asintomáticos y el diagnóstico ofrece dificultades con otras arbovirosis. El control de vectores en España es prioritario, dada la existencia de Aedes albopictus (mosquito tigre. También se recomienda el diagnóstico precoz, evitar viajes a zonas endémicas, mantener relaciones sexuales protegidas y procurar que la prioridad política, que puede evitar que esta epidemia se convierta en una enfermedad endémica de alta prevalencia, no nos haga olvidar otros problemas de salud.

  8. Evaluation of noni (Morinda citrifolia volatile profile by dynamic headspace and gas chromatography-mass spectrometry Avaliação do perfil de voláteis em noni (Morinda citrifolia por headspace dinâmico e cromatografia gasosa-espectrometria de massas

    Directory of Open Access Journals (Sweden)

    Adriana Sousa

    2010-09-01

    Full Text Available Noni is a fruit that has interested the scientific community due to its medicinal and functional activities. Different products that contain noni are already in the market, but their consumption could be impaired by their distinctive unpleasant aroma and flavor. The aim of this work was to evaluate the noni pulp volatile profile by dynamic headspace and gas chromatography-mass spectrometry. Thirty seven volatile compounds were detected, mainly alcohols (63.3%, esters (26.9%, cetones (7.4%, and acids (1.2%.O noni é um fruto que tem interessado à comunidade científica por sua atividade funcional e medicinal. Já se encontram no mercado diferentes produtos que contêm noni em sua composição, mas seu consumo tem sido prejudicado por seu aroma e sabor desagradáveis. O objetivo deste trabalho foi o de avaliar o perfil de voláteis da polpa de noni pela técnica de headspace dinâmico e cromatografia gasosa-espectrometria de massas. Foram detectados 37 compostos voláteis, sendo os principais: alcoóis (63,3%, ésteres (26,9%, cetonas (7,4% e ácidos (1,2%.

  9. Scientific publications about DNA structure-function and PCR technique in Costa Rica: A historic view (1953-2003

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    Federico J Albertazzi

    2004-09-01

    Full Text Available The spreading of knowledge depends on the access to the information and its immediate use. Models are useful to explain specific phenomena. The scientific community accepts some models in Biology after a period of time, once it has evidence to support it. The model of the structure and function of the DNA proposed by Watson & Crick (1953 was not the exception, since a few years later the DNA model was finally accepted. In Costa Rica, DNA function was first mentioned in 1970, in the magazine Biología Tropical (Tropical Biology Magazine, more than 15 years after its first publication in a scientific journal. An opposite situation occurs with technical innovations. If the efficiency of a new scientific technique is proved in a compelling way, then the acceptance by the community comes swiftly. This was the case of the polymerase chain reaction, or PCR. The first PCR machine in Costa Rica arrived in 1991, only three years after its publication. Rev. Biol. Trop. 52(3: 417-421. Epub 2004 Dic 15.La diseminación del conocimiento depende de la disponibilidad de la información y aplicar dicha información para resolver una problema. Los modelos sirvan para explicar fenómenos determinados. En Biología los modelos son aceptados por la comunidad científica después de cierto tiempo si ha probado su validez y reconocido la evidencia para apoyar dicho modelo. El modelo estructural y función de la molécula de ADN propuesto por Watson y Crick (1953 no fue la excepción pues tardó varios años en ser completamente aceptado por la comunidad científica. En Costa Rica la primera publicación relacionada con la función del ADN fue en la Revista Biología Tropical fue en 1970, más de 15 años después de ser propuesta. La situación contraria se presenta cuando son innovaciones técnicas. Si la eficiencia es demostrada, rápidamente se incorpora dentro de la comunidad. Este fue el caso de la reacción en cadena de la polimerasa, abreviado en inglés como

  10. Design and optimization of reverse-transcription quantitative PCR experiments.

    Science.gov (United States)

    Tichopad, Ales; Kitchen, Rob; Riedmaier, Irmgard; Becker, Christiane; Ståhlberg, Anders; Kubista, Mikael

    2009-10-01

    Quantitative PCR (qPCR) is a valuable technique for accurately and reliably profiling and quantifying gene expression. Typically, samples obtained from the organism of study have to be processed via several preparative steps before qPCR. We estimated the errors of sample withdrawal and extraction, reverse transcription (RT), and qPCR that are introduced into measurements of mRNA concentrations. We performed hierarchically arranged experiments with 3 animals, 3 samples, 3 RT reactions, and 3 qPCRs and quantified the expression of several genes in solid tissue, blood, cell culture, and single cells. A nested ANOVA design was used to model the experiments, and relative and absolute errors were calculated with this model for each processing level in the hierarchical design. We found that intersubject differences became easily confounded by sample heterogeneity for single cells and solid tissue. In cell cultures and blood, the noise from the RT and qPCR steps contributed substantially to the overall error because the sampling noise was less pronounced. We recommend the use of sample replicates preferentially to any other replicates when working with solid tissue, cell cultures, and single cells, and we recommend the use of RT replicates when working with blood. We show how an optimal sampling plan can be calculated for a limited budget. .

  11. Droplet digital PCR technology promises new applications and research areas.

    Science.gov (United States)

    Manoj, P

    2016-01-01

    Digital Polymerase Chain Reaction (dPCR) is used to quantify nucleic acids and its applications are in the detection and precise quantification of low-level pathogens, rare genetic sequences, quantification of copy number variants, rare mutations and in relative gene expressions. Here the PCR is performed in large number of reaction chambers or partitions and the reaction is carried out in each partition individually. This separation allows a more reliable collection and sensitive measurement of nucleic acid. Results are calculated by counting amplified target sequence (positive droplets) and the number of partitions in which there is no amplification (negative droplets). The mean number of target sequences was calculated by Poisson Algorithm. Poisson correction compensates the presence of more than one copy of target gene in any droplets. The method provides information with accuracy and precision which is highly reproducible and less susceptible to inhibitors than qPCR. It has been demonstrated in studying variations in gene sequences, such as copy number variants and point mutations, distinguishing differences between expression of nearly identical alleles, assessment of clinically relevant genetic variations and it is routinely used for clonal amplification of samples for NGS methods. dPCR enables more reliable predictors of tumor status and patient prognosis by absolute quantitation using reference normalizations. Rare mitochondrial DNA deletions associated with a range of diseases and disorders as well as aging can be accurately detected with droplet digital PCR.

  12. Result Variation and Efficiency Kinetics in Real-Time PCR

    Directory of Open Access Journals (Sweden)

    Reza Shahsiah

    2010-10-01

    Full Text Available Fluorescent monitoring of DNA amplification is the basis of real-time PCR. Absolute quantification can be achieved using a standard curve method. The standard curve is constructed by amplifying known amounts of standards under identical conditions to that of the samples.The objective of the current study is to propose a mathematical model to assess the acceptability of PCR resulys.Four commercial standards for HCV-RNA (hepatitis C virus RNA along with 6 patient samples were measured by real-time PCR, using two different RT-PCR reagents. The standard deviation of regression (Sy,x was calculated for each group of standard and compared by F-Test. The efficiency kinetics was computed by logistic regression, c2 goodness of fit test was preformed to assess the appropriateness of the efficiency curves.Calculated efficiencies were not significantly different from the value predicted by logistic regression model. Reactions with more variation showed less stable efficiency curves, with wider range of amplification efficiencies.Amplification efficiency kinetics can be computed by fitting a logistic regression curve to the gathered fluorescent data of each reaction. This model can be employed to assess the acceptability of PCR results calculated by standard curve method.

  13. Detección de Chlamydia trachomatis en muestras de exudado endocervical por la reacción en cadena de la polimerasa Detection of Chlamydia trachomatis in samples of endocervical exudate by polymerase chain reaction

    Directory of Open Access Journals (Sweden)

    Maydelín Frontela Noda

    2002-08-01

    Full Text Available Se procesaron 59 muestras de exudado endocervical, de mujeres que asistieron a 2 clínicas de infertilidad y a consulta de regulación menstrual de Ciudad de La Habana, para evaluar el desempeño de un método de detección de Chlamydia trachomatis, basado en la reacción en cadena de la polimerasa (PCR con cebadores KL1 y KL2, específicos para el plásmido. Las muestras se ensayaron por PCR-plásmido, por cultivo de células y por otro método de PCR basado en la amplificación de una región de la proteína principal de la membrana externa (MOMP de la Chlamydia, este se utilizó como ensayo confirmatorio. Se comprobó que en 43 muestras los resultados coincidían entre el cultivo y el PCR-plásmido: 4 positivas y 39 negativas. Las 16 restantes brindaron resultados discordantes. Se les realizó un estudio de inhibición a las 8 muestras cultivo positivas/PCR-plásmido negativas y se comprobó que 2 de ellas presentaban inhibidores, cuya acción fue revertida al adicionar BSA a la mezcla de reacción. De las 8 discordantes, cultivo negativo/PCR-plásmido positivas, 5 se confirmaron como positivas después del procesamiento por PCR-MOMP. Tomando como criterio de verdadero positivo la coincidencia de al menos 2 de los 3 métodos ensayados, se obtuvo sensibilidad del 100 % y especificidad del 94% para el PCR-plásmido en comparación con el 54 y 87 %, respectivamente para el cultivo. El PCR-plásmido presentó un valor predictivo positivo de 79 % y negativo de 100 %, mientras que para el cultivo fue de 50 y 89%, respectivamente. Se demostró que el PCR- plásmido, en nuestras condiciones de laboratorio, brinda resultados confiables en el diagnóstico de la Chlamydia en muestras de exudado endocervical.59 samples of endocervical exudate from women that were seen at infertility clinics and at the consultation room of menstrual regulation, in Havana City, were processed to evaluate the performance of a method to detect Chlamydia trachomatis, based on

  14. Quantification of viable bacteria in wastewater treatment plants by using propidium monoazide combined with quantitative PCR (PMA-qPCR).

    Science.gov (United States)

    Li, Dan; Tong, Tiezheng; Zeng, Siyu; Lin, Yiwen; Wu, Shuxu; He, Miao

    2014-02-01

    The detection of viable bacteria in wastewater treatment plants (WWTPs) is very important for public health, as WWTPs are a medium with a high potential for waterborne disease transmission. The aim of this study was to use propidium monoazide (PMA) combined with the quantitative polymerase chain reaction (PMA-qPCR) to selectively detect and quantify viable bacteria cells in full-scale WWTPs in China. PMA was added to the concentrated WWTP samples at a final concentration of 100 micromol/L and the samples were incubated in the dark for 5 min, and then lighted for 4 min prior to DNA extraction and qPCR with specific primers for Escherichia coli and Enterococci, respectively. The results showed that PMA treatment removed more than 99% of DNA from non-viable cells in all the WWTP samples, while matrices in sludge samples markedly reduced the effectiveness of PMA treatment. Compared to qPCR, PMA-qPCR results were similar and highly linearly correlated to those obtained by culture assay, indicating that DNA from non-viable cells present in WWTP samples can be eliminated by PMA treatment, and that PMA-qPCR is a reliable method for detection of viable bacteria in environmental samples. This study demonstrated that PMA-qPCR is a rapid and selective detection method for viable bacteria in WWTP samples, and that WWTPs have an obvious function in removing both viable and non-viable bacteria. The results proved that PMA-qPCR is a promising detection method that has a high potential for application as a complementary method to the standard culture-based method in the future.

  15. Conservación por calor

    OpenAIRE

    Cobos García, Angel; Díaz Rubio, Olga

    2011-01-01

    Esta unidade didáctica denominada Conservación por calor forma parte da materia «Tecnoloxía do procesado de alimentos» que se impartirá no primeiro semestre do 2º curso do Grao en Nutrición Humana e Dietética. A materia estrutúrase en diferentes unidades didácticas, tratando cada unha delas as diferentes tecnoloxías de procesado dos alimentos, tanto de conservación coma de transformación. A presente unidade didáctica aborda a conservación dos alimentos por calor. Este método permite destruír ...

  16. Celulitis por cuerpo extraño

    Directory of Open Access Journals (Sweden)

    Miguel B. Carrasco Guzmán

    2016-01-01

    Full Text Available Las infecciones de la piel y el tejido celular subcutáneo surgen como un grupo importante de afecciones con una alta morbilidad en edades pediátricas, generalmente relacionada con traumatismo y cuerpos extraños. Se presenta el caso de una escolar femenina de 6 años de edad, con síntomas y signos clínicos que sugieren celulitis en el muslo derecho,  por su evolución tórpida se le realizó el estudio ultrasonográfico que confirmó el diagnóstico etiológico de una celulitis secundaria a un traumatismo, provocada por la introducción de un gran cuerpo extraño, que pasó inadvertido para a familia de la menor.

  17. Aplicación de las pruebas de PCR convencional simple y múltiple para la identificación de aislamientos de Leptospira spp. en Colombia Application of conventional and multiplex PCR assays for identification of isolates of Leptospira spp. in Colombia

    Directory of Open Access Journals (Sweden)

    Natali Moreno

    2010-12-01

    Full Text Available Debido a las dificultadas asociadas con la identificación serológica de aislamientos de Leptospira ssp, se genera gran interés en la pruebas moleculares por su poder discriminatorio, reproducibilidad y fácil interpretación. Objetivo. Aplicar y validar la prueba de PCR convencional, usando dos pares de iniciadores descritos previamente y dirigidos a los genes lipL32 (PCR simple y secY/flaB (PCR múltiple, con el fin de evaluar su aplicación para identificar especies patógenas y saprófitas de Leptospira spp. Materiales y métodos. Para la estandarización de las pruebas de PCR se usó 22 cepas de referencia internacional y 12 aislamientos colombianos. Se determinó el nivel de detección de cada pareja de iniciadores, su especificidad frente a otros microorganismos causantes de enfermedades endémicas en Colombia y su capacidad de identificar especies dentro del grupo de Leptospira. Resultados. El límite de detección de la PCR simple lipL32 fue una dilución 1:10000 y para la PCR múltiple secY/flaB fue una dilución 1:100 para el gen secY y 1:1000 para flaB. La especificidad de todos los iniciadores fue de 100%. La PCR simple lipL32, mostró amplificado específico para 21/22 cepas de referencia mientras que la PCR multiple secY/flaB lo fue para 18/22 cepas. De los 12 aislamientos colombianos, siete fueron positivos por PCR lipL32 y seis lo fueron por PCR secY/flaB. Conclusiones. Los resultados más consistentes fueron obtenidos con la PCR simple lipL32 tanto en límite de detección, especificidad y utilidad para la identificación de Leptospira spp, por lo que esta prueba es aplicable a la identificación molecular de aislamientos patógenos de Leptospira spp de diversas fuentes.Serological identification of Leptospira ssp isolates is difficult to achieve. Thus, molecular testing may be of great interest thanks to its high discrimination power, reproducibility and easy interpretation. Objective. To implement and validate conventional

  18. MIASIS CUTÁNEA POR CORDYLOBIA ANTHROPOPHAGA

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    Miriam Alkorta Gurrutxaga

    2001-01-01

    Full Text Available El incremento progresivo en el número de personas que viajan a países tropicales ha hecho que las enfermedades importadas adquieran una relevancia cada vez mayor. Las miasis (o infestaciones por larvas de moscas cutáneas se encuentran entre este tipo de enfermedades siendo especialmente frecuentes en países tropicales. A propósito de la observación de un caso de miasis cutánea masiva por Cordylobia antropophaga, que ocurrió en una mujer de 34 años de edad al volver de un viaje a Senegal, se ha efectuado una revisión de los casos de miasis cutáneas forunculoides importadas publicados en España, así como de la biología, patología, tratamiento y prevención de la miasis humana por Cordylobia anthropophaga. El caso referido, se caracterizó por la infestación con un número inusualmente elevado de larvas, no sospechándose su etiología hasta la fase final de la enfermedad. La emergencia continuada de larvas (se recogieron 91 generó en la paciente un estado de ansiedad importante. Finalmente, la eliminación de las larvas provocó una rápida mejoría de la paciente. Aunque los casos de miasis cutánea no tienen la gravedad de otras enfermedades importadas, su conocimiento es necesario desde el punto de vista preventivo, diagnóstico y terapeútico. Es importante proceder a la identificación morfológica de las larvas diferenciándolas de otro tipo de miasis con implicaciones terapéuticas diferentes.

  19. Compostos antimicrobianos produzidos por Streptomyces Spp.

    OpenAIRE

    Silva, Ingrid Reis da

    2012-01-01

    O aumento crescente de bact?rias resistentes a antibi?ticos incentiva ? pesquisa por novas subst?ncias antibacterianas. Diante disso, a sele??o de microrganismos com potencial para a produ??o de novos compostos antimicrobianos tem sido amplamente estudada. Dentre estes microrganismos uma especial aten??o ? dada aos actinomicetos que apresentam capacidade de produzir uma variedade de compostos bioativos como antibi?ticos, antif?ngicos, antitumorais entre outros compostos que pod...

  20. Amar por cartas - este inferno de amara

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    Matildes Demetrio dos Santos

    1999-10-01

    Full Text Available Este trabalho tem por objetivo estudar o amor nas Folhas caídas e nas Cartas de amor à Viscondessa da Luz, de Almeida Garrett.Seus poemas cheios de erotismo e sensua lidade, inspirados numa mulher casada, Rosa Montúrar Infante, escandalizaram a sociedade portuguesado sécu lo XIX. Suas cartas de amor, em segredo, criavam uma ficção de características místicas.

  1. Por un humor ético

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    Victor Paramo Valero

    2016-04-01

    Full Text Available Ética del humor es una obra original, abundantemente documentada, de contenido científico y filosófico, que aborda un problema de gran importancia en los actuales estudios de éticas aplicadas. La ética del humor es una nueva ética aplicada que pretende comprender el fenómeno del humor a la luz de sus implicaciones éticas. Como señala el autor, Juan Carlos Siurana, reputado experto en el ámbito de la filosofía práctica, el interés por el humor es un interés por la ética. En la obra no presenta una nueva teoría filosófica del humor –las cuales se han venido sucediendo, al menos, desde los Diálogos de Platón–, sino una nueva teoría ética, que toma al humor como objeto principal de análisis. Por tanto, la finalidad es realizar una aportación dentro del ámbito de la ética. Para ello se nutre de distintos estudios psicológicos, biológicos, fisiológicos y neurológicos sobre el humor, así como de clásicas obras de filosofía que han abordado esta cuestión.

  2. El aprendizaje visto por los estudiantes venezolanos

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    Berta Elena Barrios

    2015-01-01

    Full Text Available En el siguiente trabajo se presentan los resultados de un estudio sobre las con-cepciones del aprendizaje de un grupo de estudiantes venezolanos de educa-ción primaria y media. Se trató de replicar el instrumento y procedimiento deanálisis utilizado por los investigadores Berry y Sahlberg (1996, con el objetivode caracterizar cómo conciben el aprendizaje estudiantes venezolanos entre 11y 15 años de edad y compararlo con sus pares ingleses y finlandeses. El análi-sis de resultados se basó en los criterios propuestos por dichos autores, deno-minados Pasos para un Buen Aprendizaje. Los hallazgos indican que los estu-diantes venezolanos conciben el aprendizaje como memorizar conocimientos,básicamente escolares, enseñados por los docentes. Las concepciones deambas poblaciones se ubican en una visión pasiva del aprendizaje. Se discutela vinculación de las concepciones del aprendizaje con las prácticas de la escuela.

  3. Eritema multiforme mayor desencadenado por antimicrobianos

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    Ronaldo de Carvalho Raimundo

    2010-03-01

    Full Text Available El eritema multiforme, aparece como una enfermedad sistémica con la participación de la piel y las membranas mucosas en relación con varios factores como las infecciones bacterianas o virales, y en particular la administración de drogas, analgésicos y antibióticos en general. Se presenta un paciente masculino de 29 años de edad con eritema multiforme mayor desencadenado por antimicrobianos con la aparición de lesiones vesiculares-bulloso-ulcerosas en las regiones de los labios, encías, la lengua y la mucosa genital en tratamiento de una infección del tracto urinario con norfloxacino 400 mg por una semana. Fue realizado un tratamiento de soporte con el uso de colutorios para la higienización bucal y pomada a base de corticoide para protección de las úlceras, antihistamínicos y orientación nutricional de dieta líquida hipercalórica e hiperproteica. Este síndrome está caracterizado como un proceso eruptivo buloso agudo que compromete la calidad de vida del paciente y no hay pruebas de laboratorio específicas por lo que su diagnóstico debe estar basado en la revisión minuciosa de la anamnesis y en los hallazgos clínicos.

  4. PCR detection of Bartonella spp. in the dog

    Directory of Open Access Journals (Sweden)

    Jarmila Konvalinová

    2014-01-01

    Full Text Available Our study aimed at using PCR to identify the incidence of Bartonella spp. in blood of dogs. Altogether 286 dogs of 92 breeds aged 3 month to 17 years were tested from October 2008 to December 2009. Healthy dogs as well as dogs with various clinical symptoms of disease were included in the group. Samples were tested by polymerase chain reaction (PCR specific for the presence of Bartonella spp. Following the DNA examination in 286 dogs by PCR and subsequent sequencing, two samples were identified as Bartonella henselae (0.7%. Other species of Bartonella were not found. It was the first time in the Czech Republic when incidence of Bartonella spp. was determined in dogs.

  5. [Application of rapid PCR to authenticate medicinal snakes].

    Science.gov (United States)

    Chen, Kang; Jiang, Chao; Yuan, Yuan; Huang, Lu-Qi; Li, Man

    2014-10-01

    To obtained an accurate, rapid and efficient method for authenticate medicinal snakes listed in Chinese Pharmacopoeia (Zaocysd humnades, Bungarus multicinctus, Agkistrodon acutus), a rapid PCR method for authenticate snakes and its adulterants was established based on the classic molecular authentication methods. DNA was extracted by alkaline lysis and the specific primers were amplified by two-steps PCR amplification method. The denatured and annealing temperature and cycle numbers were optimized. When 100 x SYBR Green I was added in the PCR product, strong green fluorescence was visualized under 365 nm UV whereas adulterants without. The whole process can complete in 30-45 minutes. The established method provides the technical support for authentication of the snakes on field.

  6. RUCS: Rapid identification of PCR primers for unique core sequences

    DEFF Research Database (Denmark)

    Thomsen, Martin Christen Frølund; Hasman, Henrik; Westh, Henrik

    2017-01-01

    Designing PCR primers to target a specific selection of whole genome sequenced strains can be a long, arduous, and sometimes impractical task. Such tasks would benefit greatly from an automated tool to both identify unique targets, and to validate the vast number of potential primer pairs...... for the targets in silico . Here we present RUCS, a program that will find PCR primer pairs and probes for the unique core sequences of a positive genome dataset complement to a negative genome dataset. The resulting primer pairs and probes are in addition to simple selection also validated through a complex...... in silico PCR simulation. We compared our method, which identifies the unique core sequences, against an existing tool called ssGeneFinder, and found that our method was 6.5-20 times more sensitive. We used RUCS to design primer pairs that would target a set of genomes known to contain the mcr-1 colistin...

  7. Analysis of Multiallelic CNVs by Emulsion Haplotype Fusion PCR.

    Science.gov (United States)

    Tyson, Jess; Armour, John A L

    2017-01-01

    Emulsion-fusion PCR recovers long-range sequence information by combining products in cis from individual genomic DNA molecules. Emulsion droplets act as very numerous small reaction chambers in which different PCR products from a single genomic DNA molecule are condensed into short joint products, to unite sequences in cis from widely separated genomic sites. These products can therefore provide information about the arrangement of sequences and variants at a larger scale than established long-read sequencing methods. The method has been useful in defining the phase of variants in haplotypes, the typing of inversions, and determining the configuration of sequence variants in multiallelic CNVs. In this description we outline the rationale for the application of emulsion-fusion PCR methods to the analysis of multiallelic CNVs, and give practical details for our own implementation of the method in that context.

  8. Typing DNA profiles from previously enhanced fingerprints using direct PCR.

    Science.gov (United States)

    Templeton, Jennifer E L; Taylor, Duncan; Handt, Oliva; Linacre, Adrian

    2017-07-01

    Fingermarks are a source of human identification both through the ridge patterns and DNA profiling. Typing nuclear STR DNA markers from previously enhanced fingermarks provides an alternative method of utilising the limited fingermark deposit that can be left behind during a criminal act. Dusting with fingerprint powders is a standard method used in classical fingermark enhancement and can affect DNA data. The ability to generate informative DNA profiles from powdered fingerprints using direct PCR swabs was investigated. Direct PCR was used as the opportunity to generate usable DNA profiles after performing any of the standard DNA extraction processes is minimal. Omitting the extraction step will, for many samples, be the key to success if there is limited sample DNA. DNA profiles were generated by direct PCR from 160 fingermarks after treatment with one of the following dactyloscopic fingerprint powders: white hadonite; silver aluminium; HiFi Volcano silk black; or black magnetic fingerprint powder. This was achieved by a combination of an optimised double-swabbing technique and swab media, omission of the extraction step to minimise loss of critical low-template DNA, and additional AmpliTaq Gold ® DNA polymerase to boost the PCR. Ninety eight out of 160 samples (61%) were considered 'up-loadable' to the Australian National Criminal Investigation DNA Database (NCIDD). The method described required a minimum of working steps, equipment and reagents, and was completed within 4h. Direct PCR allows the generation of DNA profiles from enhanced prints without the need to increase PCR cycle numbers beyond manufacturer's recommendations. Particular emphasis was placed on preventing contamination by applying strict protocols and avoiding the use of previously used fingerprint brushes. Based on this extensive survey, the data provided indicate minimal effects of any of these four powders on the chance of obtaining DNA profiles from enhanced fingermarks. Copyright © 2017

  9. PCR with Paracoccidioides brasiliensis specific primers: potential use in ecological studies PCR com «primers» específicos de Paracoccidioides brasiliensis: uso potencial em estudos ecológicos

    Directory of Open Access Journals (Sweden)

    S. DÍEZ

    1999-11-01

    Full Text Available The precise microenvironment of Paracoccidioides brasiliensis has not yet been discovered perhaps because the methods used are not sensitive enough. We applied to this purpose the polymerase chain reaction (PCR using three sets of specific primers corresponding to two P. brasiliensis genes. This fungus as well as several other fungi, were grown and their DNA obtained by mechanical disruption and a phenol chloroform isoamylalcohol-based purification method. The DNA served for a PCR reaction that employed specific primers from two P. brasiliensis genes that codify for antigenic proteins, namely, the 27 kDa and the 43 kDa. The lowest detection range for the 27 kDa gene was 3 pg. The amplification for both genes was positive only with DNA from P. brasiliensis; additionally, the mRNA for the 27 kDa gene was present only in P. brasiliensis, as indicated by the Northern analysis. The standardization of PCR technology permitted the amplification of P. brasiliensis DNA in artificially contaminated soils and in tissues of armadillos naturally infected with the fungus. These results indicate that PCR technology could play an important role in the search for P. brasiliensis’ habitat and could also be used in other ecological studies.O microambiente adequado do Paracoccidioides brasiliensis não foi ainda bem esclarecido, talvez porque os métodos utilizados não sejam suficientemente sensíveis. Aplicamos com este propósito, a reação em cadeia da polimerase (PCR usando três jogos de primers específicos do P. brasiliensis, correspondendo a dois dos genes do P. brasiliensis. Este fungo, assim como outros fungos, foram cultivados e seus DNAs obtidos por ruptura mecânica e purificados com mistura de fenol-clorofórmio com álcool isoamílico. Os DNAs serviram para a reação de PCR utilizando-se primers específicos para dois dos genes do P. brasiliensis que codificam para as proteínas antigênicas, denominadas, 27 kDa e 43 kDa. O limite mínimo de

  10. Soil Baiting, Rapid PCR Assay and Quantitative Real Time PCR to Diagnose Late Blight of Potato in Quarantine Programs

    Directory of Open Access Journals (Sweden)

    Touseef Hussain

    2018-05-01

    Full Text Available Phytophthora infestans (mont de Bary is a pathogen of great concern across the globe, and accurate detection is an important component in responding to the outbreaks of potential disease. Although the molecular diagnostic protocol used in regulatory programs has been evaluated but till date methods implying direct comparison has rarely used. In this study, a known area soil samples from potato fields where light blight appear every year (both A1 and A2 mating type was assayed by soil bait method, PCR assay detection and quantification of the inoculums. Suspected disease symptoms appeared on bait tubers were further confirmed by rapid PCR, inoculums were quantified through Real Time PCR, which confirms presence of P. infestans. These diagnostic methods can be highly correlated with one another. Potato tuber baiting increased the sensitivity of the assay compared with direct extraction of DNA from tuber and soil samples. Our study determines diagnostic sensitivity and specificity of the assays to determine the performance of each method. Overall, molecular techniques based on different types of PCR amplification and Real-time PCR can lead to high throughput, faster and more accurate detection method which can be used in quarantine programmes in potato industry and diagnostic laboratory.

  11. Droplet Digital™ PCR Next-Generation Sequencing Library QC Assay.

    Science.gov (United States)

    Heredia, Nicholas J

    2018-01-01

    Digital PCR is a valuable tool to quantify next-generation sequencing (NGS) libraries precisely and accurately. Accurately quantifying NGS libraries enable accurate loading of the libraries on to the sequencer and thus improve sequencing performance by reducing under and overloading error. Accurate quantification also benefits users by enabling uniform loading of indexed/barcoded libraries which in turn greatly improves sequencing uniformity of the indexed/barcoded samples. The advantages gained by employing the Droplet Digital PCR (ddPCR™) library QC assay includes the precise and accurate quantification in addition to size quality assessment, enabling users to QC their sequencing libraries with confidence.

  12. Single base pair mutation analysis by PNA directed PCR clamping

    DEFF Research Database (Denmark)

    Ørum, H.; Nielsen, P.E.; Egholm, M.

    1993-01-01

    A novel method that allows direct analysis of single base mutation by the polymerase chain reaction (PCR) is described. The method utilizes the finding that PNAs (peptide nucleic acids) recognize and bind to their complementary nucleic acid sequences with higher thermal stability and specificity...... allows selective amplification/suppression of target sequences that differ by only one base pair. Finally we show that PNAs can be designed in such a way that blockage can be accomplished when the PNA target sequence is located between the PCR primers....

  13. Identification of Meat Species by Polymerase Chain Reaction (PCR) Technique

    OpenAIRE

    İLHAK, O. İrfan; ARSLAN, Ali

    2014-01-01

    The origin of horse, dog, cat, bovine, sheep, porcine, and goat meat was determined by the polymerase chain reaction (PCR) technique, using species-specific primers. Test mixtures of meat were prepared by adding 5%, 2.5%, 1%, 0.5%, and 0.1% levels of pork, horse, cat, or dog meat to beef, sheep, and goat meat. Samples taken from those combinations were analyzed by PCR for species determination. Mitochondrial DNA (mt DNA) fragments of 439, 322, 274, 271, 225, 212, and 157 bp for horse, dog, ca...

  14. Typing of Y chromosome SNPs with multiplex PCR methods

    DEFF Research Database (Denmark)

    Sanchez Sanchez, Juan Jose; Børsting, Claus; Morling, Niels

    2005-01-01

    We describe a method for the simultaneous typing of Y-chromosome single nucleotide polymorphism (SNP) markers by means of multiplex polymerase chain reaction (PCR) strategies that allow the detection of 35 Y chromosome SNPs on 25 amplicons from 100 to 200 pg of chromosomal deoxyribonucleic acid...... factors for the creation of larger SNP typing PCR multiplexes include careful selection of primers for the primary amplification and the SBE reaction, use of DNA primers with homogenous composition, and balancing the primer concentrations for both the amplification and the SBE reactions....

  15. Detection of hepatitis C virus RNA using reverse transcription PCR

    International Nuclear Information System (INIS)

    Yap, S.F.

    1998-01-01

    Detection of the viral genome (HCV RNA) is by a combination of cDNA synthesis and PCR followed by gel analysis and/or hybridization assay. In principle, cDNA is synthesized using the viral RNA as template and the enzyme, reverse transcriptase. The cDNA is then amplified by PCR and the product detected. Agarose gel electrophoresis provides a rapid and simple detection method; however, it is non-quantitative. The assay protocol described in this paper is adapted from that published by Chan et al. Comments on various aspects of the assay are based on experience with the method in our laboratory

  16. Fecal specimens preparation methods for PCR diagnosis of human taeniosis

    Directory of Open Access Journals (Sweden)

    Nunes Cáris Maroni

    2006-01-01

    Full Text Available Sample preparation and DNA extraction protocols for DNA amplification by PCR, which can be applied in human fecal samples for taeniasis diagnosis, are described. DNA extracted from fecal specimens with phenol/chloroform/isoamilic alcohol and DNAzol® reagent had to be first purified to generate fragments of 170 pb and 600 pb by HDP2-PCR. This purification step was not necessary with the use of QIAmp DNA stool mini kit®. Best DNA extraction results were achieved after eggs disruption with glass beads, either with phenol/chloroform/isoamilic alcohol, DNAzol® reagent or QIAmp DNA stool mini kit®.

  17. Corrosión de dispositivos electrónicos por contaminantes atmosféricos en interiores de plantas industriales de ambientes áridos y marinos

    Directory of Open Access Journals (Sweden)

    Gustavo López Badilla

    2010-01-01

    Full Text Available La industria electrónica ha crecido en los últimos cincuenta años, sobre todo en los países desarrollados, contribuyendo a su progreso económico. Particularmente en el Estado de Baja California ubicada en el noroeste de México, estas empresas han prosperado en los parques industriales de Mexicali considerada como una zona árida y Ensenada, un puerto y ciudad en el Océano Pacífico, que es una región marina. En ambos ambientes, durante el invierno y el verano los principales factores climáticos en ambientes de interiores son la humedad y temperatura, que aunados a los contaminantes del aire generan corrosión en dispositivos y equipos electrónicos, disminuyendo su rendimiento operativo. El cambio de clima en interiores de plantas industriales se debe a la variación de humedad, temperatura, radiación solar, así como a la concentración de contaminantes atmosféricos como el CO, SO2, H2S, NOX, O3 y partículas sólidas PM2.5 y PM10 provenientes de exteriores de la industria electrónica. Los gases y partículas contaminantes del aire son detectados por Estaciones de Monitoreo Ambiental (EMA en Mexicali, mientras, que el SOX y Cl- se determinaron en Ensenada por la técnica de platos de sulfatación (TPS y el método de la vela húmeda (MVH. Las probetas metálicas en ambas ciudades fueron analizadas por microscopia de barrido por electrones (MBE y espectroscopia de electrones Auger (EEA para determinar los productos de corrosión. Los equipos electrónicos instalados en las plantas están constituidos por componentes de cobre, siendo un metal muy utilizado por su buena conductividad eléctrica y térmica. Debido a que están expuestos a una amplia gama de ambientes agresivos, se origina deterioro del cobre, generando fallas en los equipos y con ello pérdidas económicas. Los materiales metálicos utilizados en los dispositivos electrónicos son susceptibles a la corrosión en interiores de plantas industriales por la variaciones de

  18. Análise de citocinas pela RT-PCR em pacientes com rinite alérgica RT-PCR cytokine study in patients with allergic rhinitis

    Directory of Open Access Journals (Sweden)

    Tarcimara Moreira da Silva

    2009-02-01

    Full Text Available Rinite alérgica é uma doença que decorre de um processo inflamatório da mucosa nasal conseqüente à reação de hipersensibilidade a alérgenos inalatórios e, eventualmente, alimentares. É mediada por IgE, envolvendo diferentes células, mediadores e citocinas. OBJETIVO: Avaliar as transcrições para as seguintes citocinas: IL-4, IL-5, IL-8 e IFN-gama, particularmente importantes no processo alérgico nasal, principalmente IL-4 e IL-5. Neste estudo, optou-se por avaliar os pacientes atópicos fora das crises alérgicas, com a finalidade de se conhecer as expressões das citocinas neste período. MATERIAL E MÉTODO: Realizou-se um estudo transversal e prospectivo, selecionando-se 30 pacientes, sendo 13 pacientes portadores de rinite alérgica paucissintomáticos e 17 pacientes não-atópicos. Os grupos foram selecionados através da história, do exame clínico otorrinolaringológico e do teste alérgico cutâneo - Prick Test. O perfil das citocinas foi pesquisado nos fragmentos de mucosa nasal, através da RT-PCR semiquantitativa, escolhida por apresentar boa reprodutibilidade e especificidade, utilizando-se como referência o gene da Beta-actina. RESULTADOS: Os valores de IL-5, IL-8, IFN-gama mantiveram-se homogêneos em relação ao grupo controle. A IL-4 apresentou diferença com significância estatística. CONCLUSÃO: Os pacientes alérgicos paucissintomáticos apresentaram normalização da expressão das citocinas na mucosa nasal à exceção de IL-4.Allergic rhinitis is an inflammatory reaction of the nasal mucosa, in consequence of an IgE mediated hypersensitive reaction to inhaling allergens, involving different mediators and cytokine cells. AIM: The purpose of this study was to evaluate the transcriptions for IL-4, IL-5, IL-8 and IFN-gama, particularly important in the nasal allergy process, especially IL-4 and IL-5. For this study we decided to evaluate atopic patients who were free from allergic crises, with the purpose of

  19. Gold nanoparticle-based RT-PCR and real-time quantitative RT-PCR assays for detection of Japanese encephalitis virus

    International Nuclear Information System (INIS)

    Huang, S-H; Tsai, M-H; Lin, C-W; Yang, T-C; Chuang, P-H; Tsai, I-S; Lu, H-C; Wan Lei; Lin, Y-J; Lai, C-H

    2008-01-01

    Virus isolation and antibody detection are routinely used for diagnosis of Japanese encephalitis virus (JEV) infection, but the low level of transient viremia in some JE patients makes JEV isolation from clinical and surveillance samples very difficult. We describe the use of gold nanoparticle-based RT-PCR and real-time quantitative RT-PCR assays for detection of JEV from its RNA genome. We tested the effect of gold nanoparticles on four different PCR systems, including conventional PCR, reverse-transcription PCR (RT-PCR), and SYBR green real-time PCR and RT-PCR assays for diagnosis in the acute phase of JEV infection. Gold nanoparticles increased the amplification yield of the PCR product and shortened the PCR time compared to the conventional reaction. In addition, nanogold-based real-time RT-PCR showed a linear relationship between Ct and template amount using ten-fold dilutions of JEV. The nanogold-based RT-PCR and real-time quantitative RT-PCR assays were able to detect low levels (1-10 000 copies) of the JEV RNA genomes extracted from culture medium or whole blood, providing early diagnostic tools for the detection of low-level viremia in the acute-phase infection. The assays described here were simple, sensitive, and rapid approaches for detection and quantitation of JEV in tissue cultured samples as well as clinical samples

  20. Structural Insights into the PorK and PorN Components of the Porphyromonas gingivalis Type IX Secretion System.

    Science.gov (United States)

    Gorasia, Dhana G; Veith, Paul D; Hanssen, Eric G; Glew, Michelle D; Sato, Keiko; Yukitake, Hideharu; Nakayama, Koji; Reynolds, Eric C

    2016-08-01

    The type IX secretion system (T9SS) has been recently discovered and is specific to Bacteroidetes species. Porphyromonas gingivalis, a keystone pathogen for periodontitis, utilizes the T9SS to transport many proteins including the gingipain virulence factors across the outer membrane and attach them to the cell surface via a sortase-like mechanism. At least 11 proteins have been identified as components of the T9SS including PorK, PorL, PorM, PorN and PorP, however the precise roles of most of these proteins have not been elucidated and the structural organization of these components is unknown. In this study, we purified PorK and PorN complexes from P. gingivalis and using electron microscopy we have shown that PorN and the PorK lipoprotein interact to form a 50 nm diameter ring-shaped structure containing approximately 32-36 subunits of each protein. The formation of these rings was dependent on both PorK and PorN, but was independent of PorL, PorM and PorP. PorL and PorM were found to form a separate stable complex. PorK and PorN were protected from proteinase K cleavage when present in undisrupted cells, but were rapidly degraded when the cells were lysed, which together with bioinformatic analyses suggests that these proteins are exposed in the periplasm and anchored to the outer membrane via the PorK lipid. Chemical cross-linking and mass spectrometry analyses confirmed the interaction between PorK and PorN and further revealed that they interact with the PG0189 outer membrane protein. Furthermore, we established that PorN was required for the stable expression of PorK, PorL and PorM. Collectively, these results suggest that the ring-shaped PorK/N complex may form part of the secretion channel of the T9SS. This is the first report showing the structural organization of any T9SS component.

  1. Structural Insights into the PorK and PorN Components of the Porphyromonas gingivalis Type IX Secretion System.

    Directory of Open Access Journals (Sweden)

    Dhana G Gorasia

    2016-08-01

    Full Text Available The type IX secretion system (T9SS has been recently discovered and is specific to Bacteroidetes species. Porphyromonas gingivalis, a keystone pathogen for periodontitis, utilizes the T9SS to transport many proteins including the gingipain virulence factors across the outer membrane and attach them to the cell surface via a sortase-like mechanism. At least 11 proteins have been identified as components of the T9SS including PorK, PorL, PorM, PorN and PorP, however the precise roles of most of these proteins have not been elucidated and the structural organization of these components is unknown. In this study, we purified PorK and PorN complexes from P. gingivalis and using electron microscopy we have shown that PorN and the PorK lipoprotein interact to form a 50 nm diameter ring-shaped structure containing approximately 32-36 subunits of each protein. The formation of these rings was dependent on both PorK and PorN, but was independent of PorL, PorM and PorP. PorL and PorM were found to form a separate stable complex. PorK and PorN were protected from proteinase K cleavage when present in undisrupted cells, but were rapidly degraded when the cells were lysed, which together with bioinformatic analyses suggests that these proteins are exposed in the periplasm and anchored to the outer membrane via the PorK lipid. Chemical cross-linking and mass spectrometry analyses confirmed the interaction between PorK and PorN and further revealed that they interact with the PG0189 outer membrane protein. Furthermore, we established that PorN was required for the stable expression of PorK, PorL and PorM. Collectively, these results suggest that the ring-shaped PorK/N complex may form part of the secretion channel of the T9SS. This is the first report showing the structural organization of any T9SS component.

  2. Serosurvey for tick-borne diseases in dogs from the Eastern Amazon, Brazil Pesquisa Sorológica por doenças transmitidas por carrapatos em cães da Amazônia oriental, Brasil

    Directory of Open Access Journals (Sweden)

    Mariana Granziera Spolidorio

    Full Text Available Canine ehrlichiosis and babesiosis are the most prevalent tick-borne diseases in Brazilian dogs. Few studies have focused attention in surveying tick-borne diseases in the Brazilian Amazon region. A total of 129 blood samples were collected from dogs living in the Brazilian eastern Amazon. Seventy-two samples from dogs from rural areas of 19 municipalities and 57 samples from urban stray dogs from Santarém municipality were collected. Serum samples were submitted to Indirect Immunofluorescence Assay (IFA with antigens of Babesia canis vogeli, Ehrlichia canis, and six Rickettsia species. The frequency of dogs containing anti-B. canis vogeli, anti-E. canis, and anti-Rickettsia spp. antibodies was 42.6%, 16.2%, and 31.7%, respectively. Anti-B. canis vogeli antibodies were detected in 59.6% of the urban dogs, and in 29.1% of the rural dogs (P Ehrliquiose canina e babesiose canina são as doenças parasitárias transmitidas por carrapatos de maior prevalência em cães do Brasil. Poucos estudos pesquisaram doenças transmitidas por carrapatos na região da Amazônia brasileira. Um total de 129 amostras de sangue foram colhidas de cães da Amazônia oriental brasileira. Setenta e dois cães eram de áreas rurais de 19 municípios do Estado do Pará, e 57 amostras foram colhidas de cães errantes vadios da área urbana do município de Santarém-PA. As amostras de soro foram submetidas ao ensaio de imunofluorescência indireta, com antígenos de Babesia canis vogeli, Ehrlichia canis, e seis espécies de Rickettsia. A frequência de cães com anticorpos anti-B. canis vogeli, anti-E. canis, e anti-Rickettsia spp. foi de 42,6%, 16,2% e 31,7%, respectivamente. Anticorpos anti-B. canis vogeli foram detectados em 59,6% dos cães urbanos, e em 29,1% dos cães rurais (P < 0.05. Para E. canis, a soroprevalência foi parecida entre os cães urbanos (15,7% e rurais (16,6%. Para Rickettsia spp., cães rurais apresentaram prevalência (P < 0.05 significativamente

  3. Prevalência da infecção por Helicobacter pylori e de parasitoses intestinais em crianças do Parque Indígena do Xingu

    Directory of Open Access Journals (Sweden)

    Mario Luis Escobar-Pardo

    2011-10-01

    Full Text Available OBJETIVO: Avaliar a prevalência da infecção por Helicobacter pylori e sua associação com parasitoses intestinais em crianças da comunidade indígena do Parque Indígena do Xingu. MÉTODOS: Foram incluídas 245 crianças indígenas entre 2 e 9 anos, de seis aldeias da região do rio Xingu, afluente do Amazonas. H. pylori foi detectado pelo teste respiratório com ureia-13C. Foram coletadas amostras de ar expirado, em jejum e 30 minutos após a ingestão de 50 mg de ureia-13C diluída em 100 mL de água aromatizada com suco de maracujá. Foram coletadas amostras de fezes de 202/245 (82,4% crianças para exame protoparasitológico. RESULTADOS: A prevalência do H. pylori foi de 73,5%. Foi observada associação significativa do H. pylori com maior idade entre as diferentes aldeias e etnias. Resultaram positivas para a presença de parasitas 97,5% (198/202 das amostras de fezes, sem associação com a infecção por H. pylori. Encontrou-se, na análise multivariada, uma relação entre a infecção por giárdia e o H. pylori. As etnias Kisêjê [odds ratio (OR = 3,36] e Kaibi (OR = 4,00, e as aldeias Tuiararé (OR = 8,10, Ngojwere (OR = 4,10, Capivara (OR = 4,88, Diauarum (OR = 1,85 e Pavuru (OR = 1,40 foram fatores de risco para a infecção por H. pylori. CONCLUSÕES: Foi encontrada alta prevalência de H. pylori e de parasitose intestinal em crianças nas comunidades presentemente investigadas. No entanto, houve diferença significativa na prevalência do H. pylori entre as diversas aldeias estudadas. Verificou-se associação entre a presença de giárdia e a infecção por H. pylori.

  4. Designing multiplex PCR system of Campylobacter jejuni for efficient typing by improving monoplex PCR binary typing method.

    Science.gov (United States)

    Yamada, Kazuhiro; Ibata, Ami; Suzuki, Masahiro; Matsumoto, Masakado; Yamashita, Teruo; Minagawa, Hiroko; Kurane, Ryuichiro

    2015-01-01

    Campylobacter jejuni is responsible for the majority of Campylobacter infections. As the molecular epidemiological study of outbreaks, pulsed-field gel electrophoresis (PFGE) is performed in general. But PFGE has several problems. PCR binary typing (P-BIT) method is a typing method for Campylobacter spp. that was recently developed, and was reported to have a similar discriminatory power and stability to those of PFGE. We modified the P-BIT method from 18 monoplex PCRs to two multiplex PCR systems (mP-BIT). The same results were obtained from monoplex PCRs using original primers and multiplex PCR in the representative isolates. The mP-BIT can analyze 48 strains at a time by using 96-well PCR systems and can identify C. jejuni because mP-BIT includes C. jejuni marker. The typing of the isolates by the mP-BIT and PFGE demonstrated generally concordant results and the mP-BIT method (D = 0.980) has a similar discriminatory power to that of PFGE with SmaI digest (D = 0.975) or KpnI digest (D = 0.987) as with original article. The mP-BIT method is quick, simple and easy, and comes to be able to perform it at low cost by having become a multiplex PCR system. Therefore, the mP-BIT method with two multiplex PCR systems has high potential for a rapid first-line surveillance typing assay of C. jejuni and can be used for routine surveillance and outbreak investigations of C. jejuni in the future. Copyright © 2014 Japanese Society of Chemotherapy and The Japanese Association for Infectious Diseases. Published by Elsevier Ltd. All rights reserved.

  5. Detection and subtyping (H5 and H7) of avian type A influenza virus by reverse transcription-PCR and PCR-ELISA

    DEFF Research Database (Denmark)

    Munch, M.; Nielsen, L.P.; Handberg, Kurt

    2001-01-01

    A. A panel of reference influenza strains from various hosts including avian species, human, swine and horse were evaluated in a one tube RT-PCR using primers designed for the amplification of a 218 bp fragment of the NP gene. The PCR products were detected by PCR-ELISA by use of an internal......Avian influenza virus infections are a major cause of morbidity and rapid identification of the virus has important clinical, economical and epidemiological implications. We have developed a one-tube Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) for the rapid diagnosis of avian influenza...... catching probe confirming the NP influenza A origin. The PCR-ELISA was about 100 times more sensitive than detection of PCR products by agarose gel electrophoresis. RT-PCR and detection by PCR-ELISA is comparable in sensitivity to virus propagation in eggs. We also designed primers for the detection...

  6. One-step triplex PCR/RT-PCR to detect canine distemper virus, canine parvovirus, and canine kobuvirus.

    Science.gov (United States)

    Liu, Dafei; Liu, Fei; Guo, Dongchun; Hu, Xiaoliang; Li, Zhijie; Li, Zhigang; Ma, Jianzhang; Liu, Chunguo

    2018-01-23

    To rapidly distinguish Canine distemper virus (CDV), canine parvovirus (CPV), and canine kobuvirus (CaKoV) in practice, a one-step multiplex PCR/RT-PCR assay was developed, with detection limits of 10 2.1 TCID 50 for CDV, 10 1.9 TCID 50 for CPV and 10 3 copies for CaKoV. This method did not amplify nonspecific DNA or RNA from other canine viruses. Therefore, the assay provides a sensitive tool for the rapid clinical detection and epidemiological surveillance of CDV, CPV and CaKoV in dogs.

  7. VALIDACIÓN DE PCR PARA DETECCIÓN DE Listeria monocytogenes EN CARNES CRUDAS DE RES Y POLLO

    Directory of Open Access Journals (Sweden)

    Kirvis Torres

    2004-12-01

    Full Text Available Listeria monocytogenes es un microorganismo zoonótico emergente en la industria de alimentos, resultando degran interés para la salud pública. El objetivo de este trabajo fue validar la técnica de PCR para la detecciónde este microorganismo en carnes crudas de res y pollo. El procedimiento de extracción de ADN fue realizadocon lisozima, proteinasa K y fenol-cloroformo a partir de muestras contaminadas artificialmente. La especificidadde los cebadores LI1 y U1 fue comprobada por la amplificación de una banda de 938pb correspondiente a unfragmento del ADNr 16S; de igual manera los cebadores LF y LR amplificaron una banda del gen hlyA de750pb; lo que permitió la identificación de género (banda 938pb y especie (banda de 750pb respectivamente.Las cepas de los otros géneros bacterianos ensayados no amplificaron ninguna de las bandas específicas. Ellímite de detección para la PCR fue de 102 y 104 UFC/gr para carnes de res y pollo respectivamente; el «GoldStandard» reportó 102 UFC/ml para ambos alimentos. La comparación de la PCR vs., el método «Gold Standard»reportó en carne de pollo una concordancia observada de 98.43%, una sensibilidad del 96.9%, una especificidadde 100%, un valor predictivo positivo del 100% y un valor predictivo negativo del 97%; para la carne de restodos los parámetros anteriores fueron del 100%. Estos resultados demostraron la factibilidad de la PCR parael control de calidad de carnes crudas de res y pollo.

  8. A novel polymerase chain reaction (PCR) for rapid isolation of a new ...

    African Journals Online (AJOL)

    mediated self-formed panhandle PCR, for gene or chromosome walking. It combined the advantages of ligation-mediated PCR in its specificity and of panhandle PCR in its efficiency. Self-formed panhandle PCR was used for a new rbcS gene ...

  9. Evaluation of a PCR for detection of Actinobacillus pleuropneumoniae in mixed bacterial cultures from tonsils

    DEFF Research Database (Denmark)

    Gram, T.; Ahrens, Peter; Nielsen, J.P.

    1996-01-01

    strains of A. lignieresii. The lower detection limit of the PCR test was 10(3) A. pleuropneumoniae CFU/PCR test tube and was not affected by addition of 10(6) E. coli CFU/PCR test tube. Mixed bacterial cultures from tonsils of 101 pigs from 9 different herds were tested by culture and by PCR using four...

  10. Evaluation of the use of real-time PCR for human T cell lymphotropic virus 1 and 2 as a confirmatory test in screening for blood donors Análise do uso da PCR em tempo real para HTLV-1 e 2 como teste confirmatório na triagem de doadores de sangue

    Directory of Open Access Journals (Sweden)

    Rafaela Gomes Andrade

    2010-04-01

    Full Text Available INTRODUCTION: HTLV-1/2 screening among blood donors commonly utilizes an enzyme-linked immunosorbent assay (EIA, followed by a confirmatory method such as Western blot (WB if the EIA is positive. However, this algorithm yields a high rate of inconclusive results, and is expensive. METHODS: Two qualitative real-time PCR assays were developed to detect HTLV-1 and 2, and a total of 318 samples were tested (152 blood donors, 108 asymptomatic carriers, 26 HAM/TSP patients and 30 seronegative individuals. RESULTS: The sensitivity and specificity of PCR in comparison with WB results were 99.4% and 98.5%, respectively. PCR tests were more efficient for identifying the virus type, detecting HTLV-2 infection and defining inconclusive cases. CONCLUSIONS: Because real-time PCR is sensitive and practical and costs much less than WB, this technique can be used as a confirmatory test for HTLV in blood banks, as a replacement for WB.INTRODUÇÃO: A triagem para HTLV-1/2 em doadores de sangue geralmente utiliza imunoensaio enzimático, seguido de um método confirmatório como Western blot quando o EIA é positivo, mas este algoritmo mostra alta taxa de resultados inconclusivos, e elevado custo. MÉTODOS: Dois ensaios qualitativos de PCR em tempo real foram desenvolvidos para detectar HTLV-1 e 2 e um total de 318 amostras foram testadas por PCR (152 de doadores de sangue, 108 de portadores assintomáticos, 26 de pacientes HAM/TSP e 30 de indivíduos soronegativos. RESULTADOS: A sensibilidade e especificidade das PCR em relação aos resultados de WB foram de 99,4% e 98,5%, respectivamente. As PCR foram mais eficientes em identificar o tipo viral, a infecção pelo HTLV-2 e úteis para definir casos inconclusivos. CONCLUSÕES: Por serem sensíveis, práticas e de custo muito inferior ao do WB, as técnicas de PCR em tempo real podem ser usadas como teste confirmatório do HTLV em bancos de sangue, em substituição ao WB.

  11. Prevalência de Chlamydia trachomathis em amostras endocervicais de mulheres em São Paulo e Santa Catarina pela PCR Prevalence of Chlamydia trachomatis in endocervical samples by PCR in São Paulo and Santa Catarina

    Directory of Open Access Journals (Sweden)

    Marcos Edgar Herkenhoff

    2012-10-01

    Full Text Available INTRODUÇÃO: Nenhuma outra doença sexualmente transmissível (DST tem mostrado frequência tão elevada quanto a infecção por Chlamydia trachomatis (CT. É frequente a detecção de mulheres portadoras de danos tubários causados por esse agente, determinando infertilidade permanente e as intervenções cirúrgicas não têm demonstrado sucesso em reparar esses danos. A reação em cadeia da polimerase (PCR se mostrou mais sensível do que a cultura para a identificação de CT, principalmente em cervicite clamidiana nas mulheres. A PCR promove a detecção de sequências específicas de nucleotídeos para a CT. OBJETIVO: Analisar a prevalência de infecções causadas pela CT em mulheres nos estados de São Paulo e Santa Catarina utilizando amostras endocervicais. MATERIAIS E MÉTODOS Utilizaram-se para o presente trabalho amostras enviadas pelos laboratórios conveniados ao Genolab, pertencentes aos estados de São Paulo e de Santa Catarina. Foram consultados os resultados dos laudos de exames para CT oriundos do banco de dados do Genolab no ano de 2010. Para a obtenção e o isolamento do ácido desoxirribonucleico (DNA, utilizou-se a técnica de fenol-clorofórmio e para a amplificação do material genético, a técnica de PCR. RESULTADOS: Obteve-se uma amostra de 287 indivíduos, e desse total 56,45% das mulheres eram positivas. A amostra que obteve o maior número de positivos foi o swab endocervical, com 75%. CONCLUSÃO: As amostras biológicas provenientes do endocérvix apresentaram detecção eficiente da CT na população feminina. A alta prevalência salienta a importância no emprego do diagnóstico molecular, principalmente por este trabalho apontar esse aspecto.INTRODUCTION: No other sexually transmitted disease (STD has been as frequent as Chlamydia trachomatis (CT infection. Tubal damage caused by this agent has been frequently detected among women. This infection causes permanent infertility. Furthermore, surgical

  12. Typing of Mycoplasma pneumoniae by PCR-mediated DNA fingerprinting

    NARCIS (Netherlands)

    Ursi, D; Ieven, M; van Bever, H; Quint, W; Niesters, H G; Goossens, H

    PCR fingerprinting was used to characterize clinical isolates of Mycoplasma pneumoniae. Among 24 strains tested, two types were distinguished. Nineteen strains belonged to type 1, whereas only 5 strains belonged to type 2. The majority of strains isolated since 1991 in Belgium belong to type 1. No

  13. Halal authenticity of gelatin using species-specific PCR.

    Science.gov (United States)

    Shabani, Hessam; Mehdizadeh, Mehrangiz; Mousavi, Seyed Mohammad; Dezfouli, Ehsan Ansari; Solgi, Tara; Khodaverdi, Mahdi; Rabiei, Maryam; Rastegar, Hossein; Alebouyeh, Mahmoud

    2015-10-01

    Consumption of food products derived from porcine sources is strictly prohibited in Islam. Gelatin, mostly derived from bovine and porcine sources, has many applications in the food and pharmaceutical industries. To ensure that food products comply with halal regulations, development of valid and reliable analytical methods is very much required. In this study, a species-specific polymerase chain reaction (PCR) assay using conserved regions of mitochondrial DNA (cytochrome b gene) was performed to evaluate the halal authenticity of gelatin. After isolation of DNA from gelatin powders with known origin, conventional PCR using species-specific primers was carried out on the extracted DNA. The amplified expected PCR products of 212 and 271 bp were observed for porcine and bovine gelatin, respectively. The sensitivity of the method was tested on binary gelatin mixtures containing 0.1%, 1%, 10%, and 100% (w/w) of porcine gelatin within bovine gelatin and vice versa. Although most of the DNA is degraded due to the severe processing steps of gelatin production, the minimum level of 0.1% w/w of both porcine and bovine gelatin was detected. Moreover, eight food products labeled as containing bovine gelatin and eight capsule shells were subjected to PCR examination. The results showed that all samples contained bovine gelatin, and the absence of porcine gelatin was verified. This method of species authenticity is very useful to verify whether gelatin and gelatin-containing food products are derived from halal ingredients. Copyright © 2015 Elsevier Ltd. All rights reserved.

  14. Comparison of polymerase chain reaction (PCR) and loop-mediated ...

    African Journals Online (AJOL)

    Comparison of polymerase chain reaction (PCR) and loop-mediated isothermal amplification (LAMP) for diagnosis of Fusarium solani in human immunodeficiency virus (HIV) positive patients. ... The test was carried out in 1 h reaction at 65°C in a heater block. The specificity of the test was 100% and its sensitivity was a ...

  15. Introducing Undergraduate Students to Real-Time PCR

    Science.gov (United States)

    Hancock, Dale; Funnell, Alister; Jack, Briony; Johnston, Jill

    2010-01-01

    An experiment is conducted, which in four 3 h laboratory sessions, introduces third year undergraduate Biochemistry students to the technique of real-time PCR in a biological context. The model used is a murine erythroleukemia cell line (MEL cells). These continuously cycling, immature red blood cells, arrested at an early stage in erythropoiesis,…

  16. A Trio of Human Molecular Genetics PCR Assays

    Science.gov (United States)

    Reinking, Jeffrey L.; Waldo, Jennifer T.; Dinsmore, Jannett

    2013-01-01

    This laboratory exercise demonstrates three different analytical forms of the polymerase chain reaction (PCR) that allow students to genotype themselves at four different loci. Here, we present protocols to allow students to a) genotype a non-coding polymorphic Variable Number of Tandem Repeat (VNTR) locus on human chromosome 5 using conventional…

  17. Fuel utilization in a progressive conversion reactor (PCR)

    International Nuclear Information System (INIS)

    Leyse, C.F.; Judd, J.L.

    1981-05-01

    Preliminary studies indicate that for once-through fuel cycles, the PCR offers potential improvements over current LWRs in the following major areas: improved uranium utilization (reduced uranium demand), degraded plutonium product in spent fuel, reduced plutonium content of spent fuel, reduced amount of spent fuel, reduced fissile content of spent fuel, and reduced separative work

  18. Statistical aspects of quantitative real-time PCR experiment design

    Czech Academy of Sciences Publication Activity Database

    Kitchen, R.R.; Kubista, Mikael; Tichopád, Aleš

    2010-01-01

    Roč. 50, č. 4 (2010), s. 231-236 ISSN 1046-2023 R&D Projects: GA AV ČR IAA500520809 Institutional research plan: CEZ:AV0Z50520701 Keywords : Real-time PCR * Experiment design * Nested analysis of variance Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.527, year: 2010

  19. Design and Optimization of Reverse-Transcription Quantitative PCR Experiments

    Czech Academy of Sciences Publication Activity Database

    Tichopád, A.; Kitchen, R.; Riedmaier, I.; Becker, Ch.; Ståhlberg, A.; Kubista, Mikael

    2009-01-01

    Roč. 55, č. 10 (2009), s. 1816-1823 ISSN 0009-9147 Institutional research plan: CEZ:AV0Z50520701 Keywords : Design * optimization * RT qPCR Subject RIV: EG - Zoology Impact factor: 6.263, year: 2009

  20. Comparison of conventional culture and real-time quantitative PCR ...

    African Journals Online (AJOL)

    2009-10-28

    Oct 28, 2009 ... for each sample), which can be used to determine the success of the PCR reaction ... good performance in the absence of an internal control. First, a. GenBank query ..... the context of a health risk management programme.

  1. Bioinformatic tools and guideline for PCR primer design | Abd ...

    African Journals Online (AJOL)

    Bioinformatics has become an essential tool not only for basic research but also for applied research in biotechnology and biomedical sciences. Optimal primer sequence and appropriate primer concentration are essential for maximal specificity and efficiency of PCR. A poorly designed primer can result in little or no ...

  2. Typing of Mycoplasma pneumoniae by PCR-mediated DNA fingerprinting

    NARCIS (Netherlands)

    Ursi, D; Ieven, M; van Bever, H; Quint, W; Niesters, H G; Goossens, H

    1994-01-01

    PCR fingerprinting was used to characterize clinical isolates of Mycoplasma pneumoniae. Among 24 strains tested, two types were distinguished. Nineteen strains belonged to type 1, whereas only 5 strains belonged to type 2. The majority of strains isolated since 1991 in Belgium belong to type 1. No

  3. Real-time PCR for Strongyloides stercoralis-associated meningitis.

    Science.gov (United States)

    Nadir, Eyal; Grossman, Tamar; Ciobotaro, Pnina; Attali, Malka; Barkan, Daniel; Bardenstein, Rita; Zimhony, Oren

    2016-03-01

    Four immunocompromised patients, immigrants from Ethiopia, presented with diverse clinical manifestations of meningitis associated with Strongyloides stercoralis dissemination as determined by identification of intestinal larvae. The cerebrospinal fluid of 3 patients was tested by a validated (for stool) real-time PCR for S. stercoralis and was found positive, establishing this association. Copyright © 2016 Elsevier Inc. All rights reserved.

  4. Role of Polymerase Chain Reaction (PCR) in the detection of ...

    African Journals Online (AJOL)

    Rajeh Ali

    2014-06-20

    Jun 20, 2014 ... in 2013. Objectives: This study aimed to investigate S. aureus in some clinical samples by PCR and study .... culture, the isolates of S. aureus were incubated at 37 °C for. 18–24 h .... characteristics, and ability to form biofilm.

  5. Role of Polymerase Chain Reaction (PCR) in the detection of ...

    African Journals Online (AJOL)

    Background: Staphylococcus aureus is mainly acquired from hospital infections and demonstrated the ability of developing resistance to many antibiotics. Polymerase Chain Reaction (PCR) was used to identify antibiotic-resistant isolates. This study was conducted in Al-Mujtahed, Al-Mouwasat and the Children Hospitals in ...

  6. Polymerase Chain Reaction (PCR) provides a superior tool for the ...

    African Journals Online (AJOL)

    Polymerase Chain Reaction (PCR) provides a superior tool for the diagnosis of Pneumococcal Infection in Burkina Faso. Y Chaibou, M Congo/Ouedraogo, I Sanou, H Somlare, K Ouattara, CM Kienou, H Belem, E Sampo, SA Traore, R Traore/Ouedraogo, C Hatcher, L Mayer, X Wang, L Sangare ...

  7. Comparison of agglutination test, microscopy and nPCR for ...

    African Journals Online (AJOL)

    action (nPCR) for the detection of T. gondii infection in mice (n=399) inoculated with heart .... Direct Agglutination Test (DAT) (Toxo screen DA, Biomerieux®, France) fol- ... 0.3 µl external forward Primer (50 µM), 0.3 µl external reverse Primer.

  8. Development of a multiplex PCR assay detecting 52 autosomal SNPs

    DEFF Research Database (Denmark)

    Sanchez Sanchez, Juan Jose; Phillips, C.; Børsting, Claus

    2006-01-01

    for amplifying 52 genomic DNA fragments, each containing one SNP, in a single tube, and accurately genotyping the PCR product mixture using two single base extension reactions. This multiplex approach reduces the cost of SNP genotyping and requires as little as 0.5 ng of genomic DNA to detect 52 SNPs. We used...

  9. How useful is PCR in the diagnosis of malaria?

    NARCIS (Netherlands)

    Hänscheid, Thomas; Grobusch, Martin P.

    2002-01-01

    Polymerase chain reaction (PCR) assays are the most sensitive and specific method to detect malaria parasites, and have acknowledged value in research settings. However, the time lag between sample collection, transportation and processing, and dissemination of results back to the physician limits

  10. PCR-DGGE fingerprints of microbial successional changes during ...

    African Journals Online (AJOL)

    PCR-DGGE fingerprints of microbial successional changes during fermentation of cereal-legume weaning foods. ... African Journal of Biotechnology ... Phenotypic identification and monitoring of the dynamics of naturally occurring microbial community responsible for the spontaneous fermentation of different cereal-legume ...

  11. FTA card utility for PCR detection of Mycobacterium leprae.

    Science.gov (United States)

    Aye, Khin Saw; Matsuoka, Masanori; Kai, Masanori; Kyaw, Kyaw; Win, Aye Aye; Shwe, Mu Mu; Thein, Min; Htoo, Maung Maung; Htoon, Myo Thet

    2011-01-01

    The suitability of the FTA® elute card for the collection of slit skin smear (SSS) samples for PCR detection of Mycobacterium leprae was evaluated. A total of 192 SSS leprosy samples, of bacillary index (BI) 1 to 5, were collected from patients attending two skin clinics in Myanmar and preserved using both FTA® elute cards and 70% ethanol tubes. To compare the efficacy of PCR detection of DNA from each BI class, PCR was performed to amplify an M. leprae-specific repetitive element. Of the 192 samples, 116 FTA® elute card and 112 70% ethanol samples were PCR positive for M. leprae DNA. When correlated with BI, area under the curve (AUC) values of the respective receiver-operating characteristic curves were similar for the FTA® elute card and ethanol collection methods (AUC=0.6). Taken together, our results indicate that the FTA® elute card, which enables the collection, transport, and archiving of clinical samples, is an attractive alternative to ethanol preservation for the detection of M. leprae DNA.

  12. Pure chromosome-specific PCR libraries from single sorted chromosomes

    NARCIS (Netherlands)

    VanDevanter, D. R.; Choongkittaworn, N. M.; Dyer, K. A.; Aten, J. A.; Otto, P.; Behler, C.; Bryant, E. M.; Rabinovitch, P. S.

    1994-01-01

    Chromosome-specific DNA libraries can be very useful in molecular and cytogenetic genome mapping studies. We have developed a rapid and simple method for the generation of chromosome-specific DNA sequences that relies on polymerase chain reaction (PCR) amplification of a single flow-sorted

  13. Development of a competitive PCR assay for the quantification of ...

    African Journals Online (AJOL)

    ONOS

    2010-01-25

    Jan 25, 2010 ... quantification of total Escherichia coli DNA in water. Omar Kousar Banu, Barnard .... Thereafter the product was ligated into the pGEM®T-easy cloning ... agarose gel using the high pure PCR product purification kit. (Roche® ...

  14. QUANTITATIVE PCR OF SELECTED ASPERGILLUS, PENICILLIUM AND PAECILOMYCES SPECIES

    Science.gov (United States)

    A total of 65 quantitative PCR (QPCR) assays, incorporating fluorigenic 5' nuclease (TaqMan®) chemistry and directed at the nuclear ribosomal RNA operon, internal transcribed spacer regions (ITS1 or ITS2) was developed and tested for the detection of Aspergillus, Penicillium and ...

  15. Diagnostic PCR: Comparative sensitivity of four probe chemistries

    DEFF Research Database (Denmark)

    Josefsen, Mathilde Hartmann; Löfström, Charlotta; Sommer, Helle Mølgaard

    2009-01-01

    Three probe chemistries: locked nucleic acid (LNA), minor groove binder (MGB) and Scorpion were compared with a TaqMan probe in a validated real-time PCR assay for detection of food-borne thermotolerant Campylobacter. The LNA probe produced significantly lower Ct-values and a higher proportion of...

  16. Evaluation of nested PCR in diagnosis of fungal rhinosinusitis.

    Science.gov (United States)

    Badiee, Parisa; Gandomi, Behrooz; Sabz, Gholamabbass; Khodami, Bijan; Choopanizadeh, Maral; Jafarian, Hadis

    2015-02-01

    Given the importance of rapid diagnosis for fungal rhinosinusitis, this study aimed to evaluate the use of nested PCR to identify Aspergillus and Mucor species in clinical samples from patients with suspected fungal rhinosinusitis. Functional endoscopic sinus surgery specimens were collected from 98 patients with rhinosinusitis from 2012 to 2013. All samples were ground and cultured on sabouraud dextrose agar. The isolated fungi were identified based on their macroscopic and microscopic features. Fungal DNA was extracted from the tissue samples and nested PCR was performed with two sets of primers for Mucor and Aspergillus. Direct microscopic showed that 5.1% contained fungal components and 9.2% exhibited growth of fungi in culture. The most common agents isolated were Aspergillus fumigatus (n= 3), Aspergillus flavus (n=2), Penicillium sp (n=3) and Alternaria sp. (n=1). Mucor sp. was identified in the pathology smear from 1 patient. Positive results for fungal rhinosinusitis were obtained for a total of 10.2% by culture or pathology smear. Positive PCR results were obtained in 72 samples for Aspergillus and 31 samples for Mucor. Our results suggest that endoscopic sinus surgery specimens are not suitable for nested PCR, probably because of the accumulation of fungi that contaminate the environmental air. This drawback is a limiting factor for diagnosis with nasal cavity specimens. Therefore, molecular methods and conventional culture techniques are helpful complementary diagnostic methods to detect fungal rhinosinusitis and determine appropriate management for these patients.

  17. Quality control for quantitative PCR based on amplification compatibility test

    Czech Academy of Sciences Publication Activity Database

    Tichopád, Aleš; Bar, T.; Pecen, Ladislav; Kitchen, R.R.; Kubista, Mikael; Pfaffl, M.W.

    2010-01-01

    Roč. 50, č. 4 (2010), s. 308-312 ISSN 1046-2023 R&D Projects: GA AV ČR IAA500520809; GA AV ČR IAA500970904 Institutional research plan: CEZ:AV0Z50520701 Keywords : Quantitative PCR * Quality control * Amplification efficiency Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 4.527, year: 2010

  18. Comparison of the performance in detection of HPV infections between the high-risk HPV genotyping real time PCR and the PCR-reverse dot blot assays.

    Science.gov (United States)

    Zhang, Lahong; Dai, Yibei; Chen, Jiahuan; Hong, Liquan; Liu, Yuhua; Ke, Qiang; Chen, Yiwen; Cai, Chengsong; Liu, Xia; Chen, Zhaojun

    2018-01-01

    A new multiplex real-time PCR assay, the high-risk HPV genotyping real time PCR assay (HR HPV RT-PCR), has been developed to detect 15 high-risk HPV types with respective viral loads. In this report, a total of 684 cervical specimens from women diagnosed with vaginitis were assessed by the HR HPV RT-PCR and the PCR reaction and reverse dot blot (PCR-RDB) assays, using a PCR-sequencing method as a reference standard. A total coincidence of 97.7% between the HR HPV RT PCR and the PCR-RDB assays was determined with a Kappa value of 0.953. The HR HPV RT PCR assay had sensitivity, specificity, and concordance rates (accuracy) of 99.7%, 99.7%, and 99.7%, respectively, as confirmed by PCR-sequencing, while the PCR-RDB assay had respective rates of 98.8%, 97.1%, and 98.0%. The overall rate of HPV infection, determined by PCR-sequencing, in women diagnosed with vaginitis was 49.85%, including 36.26% of single infection and 13.6% of multiple infections. The most common infections among the 15 high-risk HPV types in women diagnosed with vaginitis were HPV-52, HPV-16, and HPV-58, with a total detection rate of 10.23%, 7.75%, and 5.85%, respectively. We conclude that the HR HPV RT PCR assay exhibits better clinical performance than the PCR-RDB assay, and is an ideal alternative method for HPV genotyping. In addition, the HR HPV RT PCR assay provides HPV DNA viral loads, and could serve as a quantitative marker in the diagnosis and treatment of single and multiple HPV infections. © 2017 Wiley Periodicals, Inc.

  19. Solid-phase PCR for rapid multiplex detection of Salmonella spp. at the subspecies level, with amplification efficiency comparable to conventional PCR

    DEFF Research Database (Denmark)

    Chin, Wai Hoe; Sun, Yi; Høgberg, Jonas

    2017-01-01

    Solid-phase PCR (SP-PCR) has attracted considerable interest in different research fields since it allows parallel DNA amplification on the surface of a solid substrate. However, the applications of SP-PCR have been hampered by the low efficiency of the solid-phase amplification. In order to incr...... diagnosis, high-throughput DNA sequencing, and single-nucleotide polymorphism analysis. Graphical abstract Schematic representation of solid-phase PCR....

  20. Vasculitis inducida por metimazol: Reporte de caso

    Directory of Open Access Journals (Sweden)

    Miguel Pinto

    2011-07-01

    Full Text Available Se reporta el caso de una paciente con enfermedad de Graves, que presentó vasculitis asociada al uso de metimazol. Mujer de 14 años, que acudió a consulta por presentar intolerancia al calor, tremor distal y palpitaciones. El examen físico mostró bocio difuso, y el perfil tiroideo, TSH suprimida y hormonas tiroideas elevadas. Los anticuerpos antiperoxidasa tiroidea fueron positivos. Se inició tratamiento con metimazol y beta bloqueadores. Después de 20 días, la paciente regresó por presentar malestar general, fiebre, poliartralgia, lesiones cutáneas maculopapulares y edema de miembros inferiores. Los anticuerpos antinucleares fueron negativos y los anticuerpos anticitoplasma de los neutrófilos (ANCA, positivos. Se suspendió el metimazol y se inició prednisona. Después de 10 días de tratamiento, las molestias desaparecieron y la paciente recibió I 131.Las vasculitis asociadas al uso de tionamidas son poco frecuentes, no dependen de la dosis y están asociadas a la presencia de anticuerpos tipo ANCA. Clásicamente, afectan a los vasos pequeños de la piel; sin embargo, también pueden afectar los riñones y pulmones. El cuadro clínico se caracteriza por artralgias y mialgias. En algunos casos puede ocurrir insuficiencia renal de grado variable. En la mayoría de casos, el cuadro remite con la suspensión de la droga; pero, en algunos se requiere el uso de glucocorticoides o inmunosupresores.(Rev Med Hered 2011;22:147-150.

  1. Cefaleia por uso excessivo de medicamentos

    Directory of Open Access Journals (Sweden)

    Ariane Maria Fonseca MIRANDA

    2015-01-01

    Full Text Available Uma variante da cefaleia crônica diária, a cefaleia por uso excessivo de medicamentos é uma manifestação clínica de frequência ≥ 15 dias por mês, durante 3 meses. Possui um diagnóstico deficiente e um tratamento dividido em etapas, sendo a desintoxicação, a etapa de fundamental importância. Este artigo apresenta uma revisão sobre o tema, contribuindo para o esclarecimento das principais manifestações clínicas, principais teorias envolvendo sua fisiopatologia e a terapêutica farmacológica empregada. A metodologia utilizada foi uma revisão de publicações europeias e americanas, no período de 2001 a 2013, nos idiomas português, inglês e espanhol. Todos os medicamentos utilizados no tratamento sintomático das cefaleias são capazes de cronificar uma cefaleia preexistente, desde que sejam utilizados excessivamente, de forma regular e continuada. A suspensão de tais agentes terapêuticos resultará em melhoria na maioria dos pacientes, porém pode ser necessária a introdução de uma terapia de suporte de transição e/ou terapia profilática. Os tratamentos nao farmacológicos, quando associados ao farmacológico, ampliam a possibilidade de resultados satisfatórios, evitando recaídas.

  2. Comparison of allele-specific PCR, created restriction-site PCR, and PCR with primer-introduced restriction analysis methods used for screening complex vertebral malformation carriers in Holstein cattle

    Science.gov (United States)

    Altınel, Ahmet

    2017-01-01

    Complex vertebral malformation (CVM) is an inherited, autosomal recessive disorder of Holstein cattle. The aim of this study was to compare sensitivity, specificity, positive and negative predictive values, accuracy, and rapidity of allele-specific polymerase chain reaction (AS-PCR), created restriction-site PCR (CRS-PCR), and PCR with primer-introduced restriction analysis (PCR-PIRA), three methods used in identification of CVM carriers in a Holstein cattle population. In order to screen for the G>T mutation in the solute carrier family 35 member A3 (SLC35A3) gene, DNA sequencing as the gold standard method was used. The prevalence of carriers and the mutant allele frequency were 3.2% and 0.016, respectively, among Holstein cattle in the Thrace region of Turkey. Among the three methods, the fastest but least accurate was AS-PCR. Although the rapidity of CRS-PCR and PCR-PIRA were nearly equal, the accuracy of PCR-PIRA was higher than that of CRS-PCR. Therefore, among the three methods, PCR-PIRA appears to be the most efficacious for screening of mutant alleles when identifying CVM carriers in a Holstein cattle population. PMID:28927256

  3. Two-temperature LATE-PCR endpoint genotyping

    Directory of Open Access Journals (Sweden)

    Reis Arthur H

    2006-12-01

    Full Text Available Abstract Background In conventional PCR, total amplicon yield becomes independent of starting template number as amplification reaches plateau and varies significantly among replicate reactions. This paper describes a strategy for reconfiguring PCR so that the signal intensity of a single fluorescent detection probe after PCR thermal cycling reflects genomic composition. The resulting method corrects for product yield variations among replicate amplification reactions, permits resolution of homozygous and heterozygous genotypes based on endpoint fluorescence signal intensities, and readily identifies imbalanced allele ratios equivalent to those arising from gene/chromosomal duplications. Furthermore, the use of only a single colored probe for genotyping enhances the multiplex detection capacity of the assay. Results Two-Temperature LATE-PCR endpoint genotyping combines Linear-After-The-Exponential (LATE-PCR (an advanced form of asymmetric PCR that efficiently generates single-stranded DNA and mismatch-tolerant probes capable of detecting allele-specific targets at high temperature and total single-stranded amplicons at a lower temperature in the same reaction. The method is demonstrated here for genotyping single-nucleotide alleles of the human HEXA gene responsible for Tay-Sachs disease and for genotyping SNP alleles near the human p53 tumor suppressor gene. In each case, the final probe signals were normalized against total single-stranded DNA generated in the same reaction. Normalization reduces the coefficient of variation among replicates from 17.22% to as little as 2.78% and permits endpoint genotyping with >99.7% accuracy. These assays are robust because they are consistent over a wide range of input DNA concentrations and give the same results regardless of how many cycles of linear amplification have elapsed. The method is also sufficiently powerful to distinguish between samples with a 1:1 ratio of two alleles from samples comprised of

  4. Intoxicación por Superwarfarinas

    OpenAIRE

    Patricia Crecente Otero; Jorge Luis Torres Triana; Ronald Macías Casanova; Luis Manuel Sánchez Rodríguez

    2012-01-01

    Las denominadas superwarfarinas se desarrollaron a partir de la década de 1970 como solución a las resistencias que habían desarrollado los roedores a los raticidas hasta entonces existentes1. Sus principales ventajas son su mayor potencia (hasta 100 veces más potentes que la warfarina)2 y el disponer de una semivida más larga (hasta 6-8 semanas). Presentamos el caso de un paciente varón que ingresa en nuestro servicio de Medicina Interna por otorragia y hematuria para estudio. Como único ant...

  5. Intoxicación ocupacional por mercurio

    OpenAIRE

    Augusto V Ramírez

    2008-01-01

    El mercurio, metal pesado ampliamente utilizado por el hombre, es muy tóxico; produce daño al sistema nervioso central, perturbaciones del comportamiento y lesiones renales. Se acumula en todos los seres vivos y no es esencial para ningún proceso biológico. La toxicidad del mercurio está directamente relacionada con su estado químico. El metilmercurio es la forma más dañina, con efectos neurotóxicos en adultos y en fetos de madres expuestas. El mercurio metálico no es menos tóxico. Las sales ...

  6. Refrigeración solar por eyector

    OpenAIRE

    Pizarro Romanillos, Raúl

    2014-01-01

    La finalidad de este trabajo es el estudio de un método de refrigeración que ayude a reducir el impacto medioambiental que producen otros sistemas de refrigeración más convencionales. Este método consiste en combinar la energía del Sol, que es renovable y gratuita, con un sistema de refrigeración por eyección que, a pesar de no tener alta eficiencia, tiene importantes ventajas como la de tener un bajo coste y una menor complejidad que otros sistemas. Para comenzar el estudio se hará un bre...

  7. Osteonecrosis de hueso maxilar inducida por bisfosfonatos

    OpenAIRE

    Vera Sempere, Francisco José

    2016-01-01

    Los bisfosfonatos son un grupo de fármacos, análogos de los pirofosfatos, utilizados en administración oral en el tratamiento de la osteoporosis, así como en formulaciones intravenosas para el tratamiento del dolor óseo y de la hipercalcemia ligada a la enfermedad tumoral metastasica (generalmente en el contexto de mieloma múltiple / cáncer de mama o próstata avanzados), actuando como un inhibidor de la reabsorción ósea, mediada por osteoclastos, así como de la apoptosis de los osteobl...

  8. Estudio del desgaste por deslizamiento en bronces

    OpenAIRE

    Carlos Holguín; John Coronado

    2007-01-01

    Se evaluaron tres bronces: SAE 67, nacional y americano usados típicamente como material de la chumacera en los molinos de caña de azúcar. Se realizaron pruebas desgaste por deslizamiento bajo la norma G77 usando bloques de bronce, anillos de acero SAE 1045 y aceite como lubricante. El tribómetro se calibró usando los anillos de acero SAE 4620 y se usó acero de herramienta SAE O1 para los bloques. Se realizaron metalografías, durezas y rugosidad de los materiales estudiados. El bronce naciona...

  9. Por uma arte no mundo da vida

    Directory of Open Access Journals (Sweden)

    Rachel Costa

    2016-12-01

    Full Text Available O artigo visa debater a análise colocada por Jacinto Lageira no texto “As razões do agir, contribuição a uma poética da ação”. O debate se estabelece no que concerne ao problema da autonomia da arte e suas consequências, tendo em vista a necessidade de pensar possibilidades contemporâneas para a experiência estética que pressuponham o mundo material. Isso é realizado a partir da teoria dos gestos do filósofo Vilém Flusser.

  10. Formação por ciclos

    OpenAIRE

    Nedbajluk,Lidia

    2006-01-01

    Artigo desenvolvido a partir dos estudos de Mestrado na Universidade Federal do Paraná, na linha de pesquisa Currículo, Conhecimento e Saberes nas Práticas Escolares, investigando a Formação por Ciclos. Analisa a diversidade de termos utilizados como complemento ao termo ciclos no ensino fundamental (Ciclos de Aprendizagem; Escola em Ciclos; Ensino em Ciclos; Ciclos de Desenvolvimento; Ciclos de Formação; Ciclo de Estudos) e esclarece a inadequação do uso dos mesmos indistintamente. Considera...

  11. Applicability of integrated cell culture reverse transcriptase quantitative PCR (ICC-RTqPCR) for the simultaneous detection of the four human enteric enterovirus species in disinfection studies

    Science.gov (United States)

    A newly developed integrated cell culture reverse transcriptase quantitative PCR (ICC-RTqPCR) method and its applicability in UV disinfection studies is described. This method utilizes a singular cell culture system coupled with four RTqPCR assays to detect infectious serotypes t...

  12. Comparison of COBAS AMPLICOR Neissefia gonorrhoeae PCR, including confirmation with N-gonorrhoeae-specific 16S rRNA PCR, with traditional culture

    NARCIS (Netherlands)

    Luijt, DS; Bos, PAJ; van Zwet, AA; Vader, PCV; Schirm, J

    A total of 3,023 clinical specimens were tested for Neisseria gonorrhoeae by using COBAS AMPLICOR (CA) PCR and confirmation of positives by N. gonorrhoeae-specific 16S rRNA PCR. The sensitivity of CA plus 16S rRNA PCR was 98.8%, compared to 68.2% for culture. Confirmation of CA positives increased

  13. Bias in the Cq value observed with hydrolysis probe based quantitative PCR can be corrected with the estimated PCR efficiency value

    NARCIS (Netherlands)

    Tuomi, Jari Michael; Voorbraak, Frans; Jones, Douglas L.; Ruijter, Jan M.

    2010-01-01

    For real-time monitoring of PCR amplification of DNA, quantitative PCR (qPCR) assays use various fluorescent reporters. DNA binding molecules and hybridization reporters (primers and probes) only fluoresce when bound to DNA and result in the non-cumulative increase in observed fluorescence.

  14. Capillary-based integrated digital PCR in picoliter droplets.

    Science.gov (United States)

    Chen, Jinyu; Luo, Zhaofeng; Li, Lin; He, Jinlong; Li, Luoquan; Zhu, Jianwei; Wu, Ping; He, Liqun

    2018-01-30

    The droplet digital polymerase chain reaction (ddPCR) is becoming more and more popular in diagnostic applications in academia and industry. In commercially available ddPCR systems, after they have been made by a generator, the droplets have to be transferred manually to modules for amplification and detection. In practice, some of the droplets (∼10%) are lost during manual transfer, leading to underestimation of the targets. In addition, the droplets are also at risk of cross-contamination during transfer. By contrast, in labs, some chip-based ddPCRs have been demonstrated where droplets always run in channels. However, the droplets easily coalesce to large ones in chips due to wall wetting as well as thermal oscillation. The loss of droplets becomes serious when such ddPCRs are applied to absolutely quantify rare mutations, such as in early diagnostics in clinical research or when measuring biological diversity at the cell level. Here, we propose a capillary-based integrated ddPCR system that is used for the first time to realize absolute quantification in this way. In this system, a HPLC T-junction is used to generate droplets and a long HPLC capillary connects the generator with both a capillary-based thermocycler and a capillary-based cytometer. The performance of the system is validated by absolute quantification of a gene specific to lung cancer (LunX). The results show that this system has very good linearity (0.9988) at concentrations ranging from NTC to 2.4 × 10 -4 copies per μL. As compared to qPCR, the all-in-one scheme is superior both in terms of the detection limit and the smaller fold changes measurement. The system of ddPCR might provide a powerful approach for clinical or academic applications where rare events are mostly considered.

  15. A Ribeiroia spp. (Class: Trematoda) - Specific PCR-based diagnostic

    Science.gov (United States)

    Reinitz, David M.; Yoshino, T.P.; Cole, Rebecca A.

    2007-01-01

    Increased reporting of amphibian malformations in North America has been noted with concern in light of reports that amphibian numbers and species are declining worldwide. Ribeiroia ondatrae has been shown to cause a variety of types of malformations in amphibians. However, little is known about the prevalence of R. ondatrae in North America. To aid in conducting field studies of Ribeiroia spp., we have developed a polymerase chain reaction (PCR)-based diagnostic. Herein, we describe the development of an accurate, rapid, simple, and cost-effective diagnostic for detection of Ribeiroia spp. infection in snails (Planorbella trivolvis). Candidate oligonucleotide primers for PCR were designed via DNA sequence analyses of multiple ribosomal internal transcribed spacer-2 regions from Ribeiroia spp. and Echinostoma spp. Comparison of consensus sequences determined from both genera identified areas of sequence potentially unique to Ribeiroia spp. The PCR reliably produced a diagnostic 290-base pair (bp) product in the presence of a wide concentration range of snail or frog DNA. Sensitivity was examined with DNA extracted from single R. ondatrae cercaria. The single-tube PCR could routinely detect less than 1 cercariae equivalent, because DNA isolated from a single cercaria could be diluted at least 1:50 and still yield a positive result via gel electrophoresis. An even more sensitive nested PCR also was developed that routinely detected 100 fg of the 290-bp fragment. The assay did not detect furcocercous cercariae of certain Schistosomatidae, Echinostoma sp., or Sphaeridiotrema globulus nor adults of Clinostomum sp. or Cyathocotyle bushiensis. Field testing of 137 P. trivolvis identified 3 positives with no overt environmental cross-reactivity, and results concurred with microscopic examinations in all cases. ?? American Society of Parasitologists 2007.

  16. Nodulose por Metotrexato Methotrexate Induced Nodulosis

    Directory of Open Access Journals (Sweden)

    Fernanda Guidolin

    2005-08-01

    Full Text Available A nodulose por metotrexato (MTX é um dos efeitos colaterais pouco conhecidos do uso desse medicamento em doses baixas. Embora classicamente descrita em casos de artrite reumatóide, tem aparecido, também, em outras doenças reumáticas. Descreve-se aqui um caso de nodulose por MTX em uma paciente com artrite reumatóide soropositiva, que utilizava esse medicamento há um ano, com bom controle do processo articular. Segue-se uma breve revisão sobre o assunto.Methotrexate-induced nodulosis is a rare side effect of this drug when it is used in low doses. Although classically described in rheumatoid arthritis patients, it may also appear in other rheumatic disorders. We describe a seropositive rheumatoid arthritis patient who developed methotrexate-induced nodulosis after using this drug for a year, with good control of articular symptoms. This case presentation is followed by a brief revision on the subject.

  17. Investigando o Consumo de Lazer por Idosos

    Directory of Open Access Journals (Sweden)

    Marcelo de Rezende Pinto

    2015-06-01

    Full Text Available A partir da constatação de que ainda são incipientes os estudos que buscam investigar a perspectiva cultural do consumo, atrelada à percepção de que pouco se pesquisou sobre o consumo de lazer de consumidores da terceira idade, o artigo tem por objetivo geral apresentar resultados de uma pesquisa empírica cujo objetivo foi investigar como as experiências de consumo nas atividades de lazer por indivíduos da terceira idade interagem com o sistema cultural e simbólico envolvido no fluxo da vida social cotidiana. Para se atingir esses objetivos, adotou-se uma perspectiva interpretativa de investigação, a partir de uma abordagem etnográfica. Os resultados parecem indicar que os processos sociais, desde a construção de relacionamentos entre familiares, amigos e vizinhos até a construção de redes de convivência com integrantes dos clubes de terceira constituem-se os principais processos presentes nas atividades de lazer desses indivíduos.

  18. Reacciones adversas por antiinflamatorios no esteroideos

    Directory of Open Access Journals (Sweden)

    Luisa Ivet Sánchez Ricardo

    2011-03-01

    Full Text Available Se realizó un estudio descriptivo y transversal en el Consultorio Médico de la Familia No. 28 perteneciente al Policlínico Universitario y Docente "Dr. Mario Muñoz Monroy" desde el 1ro. de junio al 31 de diciembre de 2008, con el objetivo de detectar las reacciones adversas más frecuentes provocadas por el consumo de antiinflamatorios no esteroideos. El universo estuvo constituido por 105 pacientes que asistieron a consulta en el tiempo de estudio y de estos se seleccionó una muestra de 60 pacientes, a quienes se les aplicó un cuestionario de preguntas donde se recogieron las variables de análisis. .Las mujeres resultaron las que más antiinflamatorios consumieron y en el rango de edades de 31-59 años. Las reacciones adversas que más se reportaron fueron la epigastralgia y la hipertensión arterial, así mismo se comprobó la automedicación en algunos pacientes.

  19. Hipotiroidismo inducido por Amiodarona: Reporte de caso

    Directory of Open Access Journals (Sweden)

    Jorge Luis Salinas Arce

    2012-03-01

    Full Text Available Se describe el caso de una mujer de 57 años de edad, con diagnóstico de síndrome de Wolf Parkinson White, en tratamiento con amiodarona 200 mg/día; que acudió por presentar palpitaciones, fatiga y aumento de sueño. Al examen físico no se encontró bocio, pero los reflejos osteotendinosos estaban prolongados. Los exámenes auxiliares mostraron TSH elevado, T4 libre disminuido y anticuerpos antiperoxidasa tiroidea negativos. Se suspendió la amiodarona y se inició levotiroxina. Después de dos meses; los niveles de TSH y T4 libre eran normales y la paciente no presentaba molestias. La amiodarona está asociada a diversos efectos adversos que pueden limitar su uso. Entre estos efectos adversos, se describe el hipotiroidismo inducido, que se caracteriza por TSH elevado, T4 libre disminuido y síntomas inespecíficos como fatiga, intolerancia al frío y piel seca. El tratamiento de elección es la levotiroxina.

  20. Application of droplet digital PCR for quantitative detection of Spiroplasma citri in comparison with real time PCR

    Science.gov (United States)

    Droplet digital Polymerase chain reaction (ddPCR) is a unique approach to measure the absolute copy number of nucleic acid targets without the need of external standards. It is a promising DNA quantification technology for medical diagnostics but there are only a few reports of its use for plant pat...

  1. Absolute quantification of olive oil DNA by droplet digital-PCR (ddPCR): Comparison of isolation and amplification methodologies.

    Science.gov (United States)

    Scollo, Francesco; Egea, Leticia A; Gentile, Alessandra; La Malfa, Stefano; Dorado, Gabriel; Hernandez, Pilar

    2016-12-15

    Olive oil is considered a premium product for its nutritional value and health benefits, and the ability to define its origin and varietal composition is a key step towards ensuring the traceability of the product. However, isolating the DNA from such a matrix is a difficult task. In this study, the quality and quantity of olive oil DNA, isolated using four different DNA isolation protocols, was evaluated using the qRT-PCR and ddPCR techniques. The results indicate that CTAB-based extraction methods were the best for unfiltered oil, while Nucleo Spin-based extraction protocols showed greater overall reproducibility. The use of both qRT-PCR and ddPCR led to the absolute quantification of the DNA copy number. The results clearly demonstrate the importance of the choice of DNA-isolation protocol, which should take into consideration the qualitative aspects of DNA and the evaluation of the amplified DNA copy number. Copyright © 2016 Elsevier Ltd. All rights reserved.

  2. Lucro por ação

    Directory of Open Access Journals (Sweden)

    Gabriel Moreira Campos

    2001-08-01

    Full Text Available Este trabalho tem por objetivo demonstrar os principais conceitos acerca do Lucro por Ação (Earnings per Share, o qual se apresenta como um quociente de grande utilidade nas entidades. Serão demonstrados aspectos sobre o assunto presentes no Brasil, bem como as normas aplicáveis nos Estados Unidos, emanadas do Financial Accounting Standards Board (FASB, e as normas internacionais, emanadas do International Accounting Standards Committee (IASC. De forma a possibilitar uma visualização mais completa dos conceitos envolvidos, serão desenvolvidos exemplos de sua aplicação. O Lucro (Resultado por Ação pode ser calculado em sua forma básica e em sua forma diluída. Na forma básica, não são considerados os efeitos dos instrumentos potencialmente dilutivos, ao passo que, no cálculo do Lucro (Resultado por Ação Diluído, são. Como instrumentos financeiros potencialmente dilutivos temos as ações preferenciais conversíveis, as debêntures conversíveis e os bônus de subscrição, que podem ser convertidos em ações ordinárias, caracterizando, assim, o próprio potencial dilutivo desses instrumentos. Dessa forma, o trabalho em questão foi dividido em três partes principais, sendo que nas duas últimas constam os referidos exemplos de cálculo do Lucro por Ação em sua forma básica e em sua forma diluída: • aspectos observados no Brasil; • normas emanadas do FASB; • normas emanadas do IASC.The objective of this paper is to demonstrate the principal concepts about Earnings per Share, which is presented as a quotient of great usefulness for the companies. The subject is presented in three parts: in the first part, we will demonstrate relevant aspects that are present in Brazil. In the second part, the applicable standards in the United States will be discussed, which are issued by the Financial Accounting Standards Board (FASB. In the third part, the international standards are dealt with, which are issued by the

  3. Progreso en el logro de los Objetivos de Desarrollo del Milenio: la mortalidad por cáncer de cérvix desciende en Colombia

    Directory of Open Access Journals (Sweden)

    Jancy A. Huertas Q

    2015-05-01

    Full Text Available Colombia cumpliendo en 2015 la fecha establecida para el alcance de los Objetivos de Desarrollo del Milenio (odm, ha logrado un descenso progresivo en las tasas de incidencia y mortalidad por cáncer de cuello uterino durante el decenio 2000 - 2010. En este período, la tasa de mortalidad descendió significativamente para las mujeres de todas las edades (11,4% en 1998 – 6,9 en 2011, meta a 2015: 6,8% y aumentó la proporción de casos in situ detectados oportunamente (63,31% en 2012. Colombia asumió el cáncer como un problema de salud pública y logró posicionarlo en la agenda pública. De igual forma, el cambio en el conocimiento y el autocuidado de la población, dieron como resultado un aumento en el pronóstico de las pacientes. A pesar de estos avances, el país continúa concentrando esfuerzos en reducir tasas de incidencia y mortalidad, aumentar los niveles de tecnología y promover mayor desarrollo en las regiones, mejorar sustancialmente el derecho de las mujeres a ser protegidas contra esta enfermedad, a través de acceso sin barreras a los programas de tamización y tratamientos del cáncer de cuello uterino. Y finalmente, la inclusión más amplia de la vacuna contra el vph con intervalo de cada 5 años, y que tiene un mayor potencial, especialmente entre las mujeres más jóvenes. La pregunta clave hoy en día es cómo acelerar ese ritmo de progreso en los indicadores propuestos por la agenda para el desarrollo después del 2015: Objetivos de Desarrollo Sostenible (ods, y ofrecer suficientes ejemplos de estrategias eficaces y adecuadas, y proporcionar experiencias en un contexto latinoamericano.

  4. Measurement of Epstein-Barr virus DNA loads in whole blood and plasma by TaqMan PCR and in peripheral blood lymphocytes by competitive PCR.

    Science.gov (United States)

    Wadowsky, Robert M; Laus, Stella; Green, Michael; Webber, Steven A; Rowe, David

    2003-11-01

    Epstein-Barr virus (EBV) DNA load values were measured in samples of whole blood (n = 60) and plasma (n = 59) by TaqMan PCR and in samples of peripheral blood lymphocytes (PBLs) (n = 60) by competitive PCR (cPCR). The samples were obtained from 44 transplant recipients. The whole-blood and PBL loads correlated highly (r(2) > 0.900), whereas the plasma and PBL loads correlated poorly (r(2) = 0.512). Testing of whole blood by TaqMan PCR is an acceptable alternative to testing of PBLs by cPCR for quantifying EBV DNA load.

  5. Frecuencia de las mutaciones más comunes del gen CFTR en pacientes peruanos con fibrosis quística mediante la técnica ARMS-PCR

    OpenAIRE

    Aquino, Ruth; Protzel, Ana; Rivera, Juan; Abarca, Hugo; Dueñas, Milagros; Nestarez, Cecilia; Purizaga, Nestor; Diringer, Benoit

    2017-01-01

    Objetivos. Determinar la frecuencia de las diez mutaciones más comúnmente reportadas en América Latina del gen CFTR mediante Sistema de Mutación Refractario a la amplificación por PCR (ARMS-PCR) en los pacientes con fibrosis quística (FQ) de dos instituciones hospitalarias de referencia en el Perú durante el año 2014. Materiales y métodos. Se evaluó la frecuencia de las diez comúnmente reportadas más comúnmente reportadas del gen CFTR en los pacientes del Hospital Nacional Edgardo Rebagliati ...

  6. Diagnosis of the fragile X syndrome in males using methylation-specific PCR of the FMRI locus

    Directory of Open Access Journals (Sweden)

    Sérgio D.J. Pena

    1999-06-01

    Full Text Available We have developed a non-isotopic technique based on methylation-specific PCR (MSP of the FMR1 promoter for the identification of fragile X full mutations among men. DNA samples were first treated with sodium bisulfite to convert unmethylated, but not methylated, cytosines to uracil, followed by PCR amplification with oligonucleotide primers specific for methylated versus unmethylated DNA. We designed two primer pairs: one produced a 142-bp fragment from the bisulfite-treated methylated CpG island, while the other generated an 84-bp product from the treated non-methylated promoter. In normal males only the 84-bp fragment was seen, while the diagnosis of FRAXA was doubly indicated by the appearance of a 142-bp product together with absence or weak visualization of the 84-bp band. As an indispensable internal control for the efficiency of the sodium bisulfite treatment, we used a primer pair specific for the imprinted maternal methylated version of the CpG island of the SNRPN gene on human chromosome 15. Using the methylation-specific PCR we identified with 100% sensitivity and accuracy eight previously diagnosed FRAXA male patients mixed with 42 normal controls. Because of its simplicity and high efficiency, methylation-specific PCR may become the method of choice for the diagnosis of the fragile X syndrome in mentally retarded males.Nós desenvolvemos uma técnica não-isotópica baseada na PCR para a identificação de mutações completas da síndrome do X-frágil em homens. O método é baseado na PCR específica para metilação do promotor do gene FMR1. Amostras de DNA são tratadas com bissulfito de sódio para converter citosinas não-metiladas para uracilo, seguindo-se amplificação por PCR com oligonucleotídeos iniciadores específicos para DNA metilado versus não-metilado. Desenhamos dois iniciadores: um produz um fragmento de 142 pb da ilha CpG metilada tratada com bissulfito de sódio, enquanto o outro gera um produto de 84 pb do

  7. Infección por virus papiloma humano en pacientes con citología de cuello uterino negativa

    OpenAIRE

    Toro de Méndez, Morelva; López de Sánchez, Mercedes

    2017-01-01

    Objetivo: Examinar la prevalencia de la infección por virus papiloma humano en pacientes con citologías de cuello uterino negativas, en un programa de pesquisa de cáncer de cuello de la Misión Barrio Adentro. Métodos: Se analizaron 3883 muestras citológicas convencionales de cuello uterino según el sistema Bethesda 2001, de las cuales 3651 (94,0 %) eran citologías normales. Se usó PCR/RFLP para la detección y tipificación de virus de papiloma humano. Resultados: La edad promedio de las pacien...

  8. PCR múltiple para la detección de los genes sea, seb, sec, sed y see de Staphylococcus aureus: Caracterización de aislamientos de origen alimentario Multiplex PCR for the detection of sea, seb, sec, sed and see genes of Staphylococcus aureus: Characterization of isolates from food

    Directory of Open Access Journals (Sweden)

    E. A. Manfredi

    2010-09-01

    Full Text Available La presencia de Staphylococcus aureus en los alimentos representa un riesgo potencial para la salud pública; sus enterotoxinas son el principal factor de virulencia. La detección de las enterotoxinas de S. aureus puede realizarse por ELISA, aunque sólo es posible detectar el pool de enterotoxinas SEA, SEB, SEC, SED y SEE. Los objetivos del presente trabajo fueron optimizar dos técnicas de PCR múltiple para la detección de los genes sea, seb, sec, sed y see de S. aureus y caracterizar un conjunto de 115 aislamientos de Staphylococcus spp. asociados a intoxicaciones alimentarias provenientes de diferentes provincias de Argentina. La caracterización se realizó por pruebas bioquímicas, ELISA y PCR. Sesenta y ocho aislamientos (59,1% fueron positivos por ELISA, mientras que 61 (53% fueron positivos por PCR. De los aislamientos positivos por PCR, 34 (55,7% portaron el gen sea, 9 (14,8% el gen seb, 5 (8,1% el gen see, 4 (6,5% el gen sec, 6 (9,9% los genes sea y seb, 2 (3,3% los genes sea y sec, y 1 (1,7% los genes sea y sed. Este es el primer estudio de caracterización genotípica de aislamientos de S. aureus asociados con brotes de intoxicación alimentaria registrados en distintas provincias argentinas.The presence of Staphylococcus aureus in food represents a potential risk to public health, being its enterotoxins the major virulence factor. Enterotoxin detection can be determined by ELISA, but only for the pool of enterotoxins SEA, SEB, SEC, SED and SEE. The main aims of this study were to optimize two PCR techniques for detection of S. aureus sea, seb, sec, sed and see, and to characterize Staphylococcus spp. isolates associated with food intoxication. Two PCR techniques were optimized and 115 Staphylococcus spp. isolates from Ciudad Autónoma de Buenos Aires, and Buenos Aires, Córdoba, and Neuquén provinces were characterized. The characterization was performed by biochemical tests, ELISA and PCR. Sixty-eight isolates (59.1% were

  9. Molecular methods (digital PCR and real-time PCR) for the quantification of low copy DNA of Phytophthora nicotianae in environmental samples.

    Science.gov (United States)

    Blaya, Josefa; Lloret, Eva; Santísima-Trinidad, Ana B; Ros, Margarita; Pascual, Jose A

    2016-04-01

    Currently, real-time polymerase chain reaction (qPCR) is the technique most often used to quantify pathogen presence. Digital PCR (dPCR) is a new technique with the potential to have a substantial impact on plant pathology research owing to its reproducibility, sensitivity and low susceptibility to inhibitors. In this study, we evaluated the feasibility of using dPCR and qPCR to quantify Phytophthora nicotianae in several background matrices, including host tissues (stems and roots) and soil samples. In spite of the low dynamic range of dPCR (3 logs compared with 7 logs for qPCR), this technique proved to have very high precision applicable at very low copy numbers. The dPCR was able to detect accurately the pathogen in all type of samples in a broad concentration range. Moreover, dPCR seems to be less susceptible to inhibitors than qPCR in plant samples. Linear regression analysis showed a high correlation between the results obtained with the two techniques in soil, stem and root samples, with R(2) = 0.873, 0.999 and 0.995 respectively. These results suggest that dPCR is a promising alternative for quantifying soil-borne pathogens in environmental samples, even in early stages of the disease. © 2015 Society of Chemical Industry.

  10. IMPLEMENTACIÓN DE LA PCR (REACCIÓN EN CADENA DE LA POLIMERASA PARA EL DIAGNÓSTICO DE LA BRUCELOSIS DE BOVINOS EN EL ECUADOR

    Directory of Open Access Journals (Sweden)

    Orly Fernando Cevallos Falquez

    2008-06-01

    Full Text Available En el presente trabajo se implementó la técnica de PCR para la detección de Brucella abortus, en muestras de sangre, en comparación con la prueba Rosa de bengala. La amplificación del ADN se realizó utilizando tres oligonucleotidos homólogos correspondientes a la secuencia 16 ARNr de Brucella abortus, dando una amplificación de 900 y 725 pb respectivamente. Un total de 172 muestras de sangre fueron recolectadas de 4 hatos con prevalencia histórica de presencia de brucelosis. Resultaron 142 negativas con la prueba de serológia y 143 con PCR, 30 resultaron positivas con la prueba serológica Rosa de bengala y 29 salieron positivas con PCR. Todos los animales que salieron positivos con Rosa de bengala, 4 presentaban síntomas clínicos como son; abortos, terneros débiles, baja producción de carne y leche. Los otros 26 animales resultaron negativos con PCR y estos animales no presentaron los síntomas clínicos. De los 142 animales que dieron negativo con Rosa de bengala, 25 resultaron positivos con PCR. Los resultados muestran que la detección de los animales positivos mediante la PCR fue más especificas y sensibles que la prueba serológica de Rosa de bengala, por lo tanto es una herramienta muy útil en el diagnóstico de Brucella abortus.

  11. Hemiplejía aguda infantil asociada a infección por enterovirus

    OpenAIRE

    Muñoz, Erika; Caramuta, Luciana; Frenkel, Susana; Cáceres, Lidia

    2005-01-01

    El ictus isquémico en la infancia es una entidad infrecuente, en el 50% de los casos no existe una causa identificable. Sin embargo, con el advenimiento de nuevas técnicas diagnósticas se han podido conocer más afecciones causales. Presentamos el caso de una paciente de 9 años con hemiplejía aguda izquierda, con estudios de neuroimágenes poco significativos y en la cual el análisis del líquido cefalorraquídeo por método de reacción en cadena de polimerasa (PCR) para ARN viral, fue positivo pa...

  12. Sporulation properties and antimicrobial susceptibility in endemic and rare Clostridium difficile PCR ribotypes.

    Science.gov (United States)

    Zidaric, Valerija; Rupnik, Maja

    2016-06-01

    Increased sporulation and antibiotic resistance have been proposed to be associated with certain Clostridium difficile epidemic strains such as PCR ribotype 027. In this study we examined these properties in another widespread PCR ribotype, 014/020, in comparison to prevalent PCR ribotype 002 and a group of rarely represented PCR ribotypes. Highest sporulation was observed in 014/020 strains at 24 h, while after 72 h PCR ribotype 002 and rare PCR ribotypes formed higher total number of spores. PCR ribotype 014/020 strains exhibited slightly higher resistance to tested antimicrobials, followed by group of rare PCR ribotypes and less common PCR ribotype 002. Neither sporulation properties nor antibiotic resistance clearly differed in endemic and rare strains. Copyright © 2016 Elsevier Ltd. All rights reserved.

  13. A multicenter study of viable PCR using propidium monoazide to detect Legionella in water samples.

    Science.gov (United States)

    Scaturro, Maria; Fontana, Stefano; Dell'eva, Italo; Helfer, Fabrizia; Marchio, Michele; Stefanetti, Maria Vittoria; Cavallaro, Mario; Miglietta, Marilena; Montagna, Maria Teresa; De Giglio, Osvalda; Cuna, Teresa; Chetti, Leonarda; Sabattini, Maria Antonietta Bucci; Carlotti, Michela; Viggiani, Mariagabriella; Stenico, Alberta; Romanin, Elisa; Bonanni, Emma; Ottaviano, Claudio; Franzin, Laura; Avanzini, Claudio; Demarie, Valerio; Corbella, Marta; Cambieri, Patrizia; Marone, Piero; Rota, Maria Cristina; Bella, Antonino; Ricci, Maria Luisa

    2016-07-01

    Legionella quantification in environmental samples is overestimated by qPCR. Combination with a viable dye, such as Propidium monoazide (PMA), could make qPCR (named then vPCR) very reliable. In this multicentre study 717 artificial water samples, spiked with fixed concentrations of Legionella and interfering bacterial flora, were analysed by qPCR, vPCR and culture and data were compared by statistical analysis. A heat-treatment at 55 °C for 10 minutes was also performed to obtain viable and not-viable bacteria. When data of vPCR were compared with those of culture and qPCR, statistical analysis showed significant differences (P 0.05). Overall this study provided a good experimental reproducibility of vPCR but also highlighted limits of PMA in the discriminating capability of dead and live bacteria, making vPCR not completely reliable. Copyright © 2016 Elsevier Inc. All rights reserved.

  14. Fluorescence quantitative PCR in detection of HBV DNA

    International Nuclear Information System (INIS)

    Shen Zheng; Li Ming; Shen Xia

    2003-01-01

    Objective: To study the relationship between the serum content of HBV-DNA and expression of serological markers with HBV infection patients. Methods: Serum samples from 375 hepatitis B patients with different clinical status and 70 normal persons were quantitated for HBV-DNA by FQ-PCR. Results: The average of HBV-DNA contents in the patient in the groups of HBsAg (+) and of HBeAg(+) were significantly higher than those in the group of HBsAg(-) and of HBeAg(-). Even in the group of HBeAg negative, high HBV-DNA contents might still be present in both the HBeAg(+) and HBeAg(-) groups. Conclusion: FQ-PCR can be used to monitor the real state of HBV infection, replication and the course of disease

  15. Identification of Tilletia species using rep-PCR fingerprinting technique

    Directory of Open Access Journals (Sweden)

    Župunski Vesna

    2011-01-01

    Full Text Available Analyzing 167 non-processed seed samples of wheat, it was found that 145 samples (86.8 % were contaminated with Tilletia species, while 22 (13.2 % samples were not contaminated. By using rep-PCR fingerprinting technique, it was found that DNA isolates of T. tritici originated from Serbian wheat samples had 80 % similarity with positive control for T. tritici. One isolate shared similarity of 60% with T. tritici, T. controversa and T. laevis. It was supposed that this isolate belongs to T. bromi. Isolate of T. laevis shared a similarity of 70 % with isolates of T. tritici and T. controversa, while T. walkeri was more than 10 % similar with T. tritici, T. controversa and T. laevis. Although T. controversa and T. tritici had high percent of genetic similarity, they were clustered separately. Our results suggest that rep-PCR fingerprinting could be a useful tool for monitoring presence of morphologically similar Tilletia species in wheat production areas.

  16. WetLab-2: Providing Quantitative PCR Capabilities on ISS

    Science.gov (United States)

    Parra, Macarena; Jung, Jimmy Kar Chuen; Almeida, Eduardo; Boone, Travis David; Schonfeld, Julie; Tran, Luan Hoang

    2015-01-01

    The objective of NASA Ames Research Centers WetLab-2 Project is to place on the ISS a system capable of conducting gene expression analysis via quantitative real-time PCR (qRT-PCR) of biological specimens sampled or cultured on orbit. The WetLab-2 system is capable of processing sample types ranging from microbial cultures to animal tissues dissected on-orbit. The project has developed a RNA preparation module that can lyse cells and extract RNA of sufficient quality and quantity for use as templates in qRT-PCR reactions. Our protocol has the advantage that it uses non-toxic chemicals, alcohols or other organics. The resulting RNA is transferred into a pipette and then dispensed into reaction tubes that contain all lyophilized reagents needed to perform qRT-PCR reactions. These reaction tubes are mounted on rotors to centrifuge the liquid to the reaction window of the tube using a cordless drill. System operations require simple and limited crew actions including syringe pushes, valve turns and pipette dispenses. The resulting process takes less than 30 min to have tubes ready for loading into the qRT-PCR unit.The project has selected a Commercial-Off-The-Shelf (COTS) qRT-PCR unit, the Cepheid SmartCycler, that will fly in its COTS configuration. The SmartCycler has a number of advantages including modular design (16 independent PCR modules), low power consumption, rapid thermal ramp times and four-color detection. The ability to detect up to four fluorescent channels will enable multiplex assays that can be used to normalize for RNA concentration and integrity, and to study multiple genes of interest in each module. The WetLab-2 system will have the capability to downlink data from the ISS to the ground after a completed run and to uplink new programs. The ability to conduct qRT-PCR on-orbit eliminates the confounding effects on gene expression of reentry stresses and shock acting on live cells and organisms or the concern of RNA degradation of fixed samples. The

  17. Targeted resequencing and variant validation using pxlence PCR assays

    Directory of Open Access Journals (Sweden)

    Frauke Coppieters

    2016-01-01

    Full Text Available The advent of next-generation sequencing technologies had a profound impact on molecular diagnostics. PCR is a popular method for target enrichment of disease gene panels. Using our proprietary primer-design pipeline, primerXL, we have created almost one million assays covering over 98% of the human exome. Here we describe the assay specification and both in silico and wet-lab validation of a selected set of 2294 assays using both next-generation sequencing and Sanger sequencing. Using a universal PCR protocol without optimization, these assays result in high coverage uniformity and limited non-specific coverage. In addition, data indicates a positive correlation between the predictive in silico specificity score and the amount of assay non-specific coverage.

  18. Automated 5 ' nuclease PCR assay for identification of Salmonella enterica

    DEFF Research Database (Denmark)

    Hoorfar, Jeffrey; Ahrens, Peter; Rådström, P.

    2000-01-01

    -point fluorescence (FAM) signals for the samples and positive control (TET) signals (relative sensitivity [Delta Rn], >0.6). The diagnostic specificity of the method was assessed using 120 non-Salmonella strains, which all resulted in negative FAM signals (Delta Rn, less than or equal to 0.5). All 100 rough...... Salmonella strains tested resulted in positive FAM and TET signals. In addition, it was found that the complete PCR mixture, predispensed in microwell plates, could be stored for up to 3 months at -20 degrees C, Thus, the diagnostic TaqMan assay developed can be a useful and simple alternative method......A simple and ready-to-go test based on a 5' nuclease (TaqMan) PCR technique was developed for identification of presumptive Salmonella enterica isolates. The results were compared with those of conventional methods. The TaqMan assay was evaluated for its ability to accurately detect 210 S. enterica...

  19. La Violencia Latinoamericana vista por los Economistas

    Directory of Open Access Journals (Sweden)

    Salama Pierre

    2003-08-01

    Full Text Available

    Salama, Pierre. "La Violencia Lationoamericana vista por los Economistas", Cuadernos de Economía, V. XXII, n. 38, Bogotá, 2003 páginas 179-198.

    Este articulo sintetiza los estudios económicos publicados recientemente sobre la violencia en América Latina y analiza las causas suceptibles a acrecentarla: pobreza, desigualdad de los ingresos en nivel y variación, impunidad, diferencia entre la probabilidad de ser arrestado y la severidad de la pena impuesta, y narcotrafico. Sin embargo, la explicación de la violencia no se limitará a los factores económicos, puesto que el autor privilegia un enfoque inspirado en la antropología y la sociología.

  20. Toxicidad por óxido de etileno

    Directory of Open Access Journals (Sweden)

    Téllez M. Jairo A.

    1993-08-01

    Full Text Available

    El óxido de etileno, es un compuesto epóxido gaseoso, ampliamente utilizado en nuestro medio como agente esterilizante y de desinfección en instituciones hospitalarias. Los estudios realizados en varios países han demostrado efectos sobre la salud humana, caracterizados por  hipersensibilidad dérmica, irritación del tracto respiratorio, irritación ocular, excitación del S.N.C. y se asocia con presencia de abortos expontáneos, aberraciones cromosómicas e irritación crónica de laringe y tráquea en personal expuesto ocupacionalmente.

  1. Infeções alimentares por Campylobacter

    OpenAIRE

    Neto, Carlota Duarte Castro

    2017-01-01

    Dissertação para obtenção do grau de Mestre no Instituto Superior de Ciências da Saúde Egas Moniz Após o isolamento bem-sucedido em fezes na década de setenta do século passado (1972), Campylobacter tornou-se rapidamente a espécie bacteriana mais comumente associada a doenças diarreicas em todo o mundo. A infeção por Campylobacter provoca um espectro de doenças, incluindo enterite aguda, infeções extra-gastrointestinais e complicações pós-infeciosas. A doença gastrointestinal autolimita...

  2. fluidizado por proceso autotérmico

    Directory of Open Access Journals (Sweden)

    ZULAMITA ZAPATA BENABITHE

    2005-01-01

    Full Text Available El presente trabajo se basa en el estudio de la producción de carbón activado a partir de carbón mineral subbituminoso en un reactor de lecho fluidizado por proceso autotérmico. El carbón utilizado provino de la región de Amagá (Antioquia - Colombia y fue activado en dos reactores de lecho fluidizado de diferente diámetro interno, 10 cm y 22 cm. Los valores del área superficial específica y el volumen de poro de las muestras activadas fueron determinados a partir de las isotermas de N2 y CO2. Los resultados experimentales mostraron que los valores de áreas superficiales BET y DR y del Índice de Yodo se encontraron alrededor de 460 m2/g (SBET, 490 m2/g (SDR y 460 mg I2/g carbón.

  3. Epidemiología molecular de infección nosocomial por Klebsiella pneumoniae productora de beta-lactamasas de espectro extendido.

    Directory of Open Access Journals (Sweden)

    Paula Andrea Espinal

    2004-09-01

    Full Text Available La epidemiología molecular aplicada al estudio de las infecciones nosocomiales ha sido fundamental para la formulación y la evaluación de las medidas de control; con este fin, se caracterizaron microbiológica y molecularmente aislamientos de Klebsiella pneumoniae productores de beta-lactamasas de espectro extendido (BLEE obtenidos de pacientes en un hospital de tercer nivel de Bogotá, D.C., Colombia. Se tipificaron quince aislamientos por electroforesis en gel de campo pulsado (PFGE y por amplificación de secuencias de ADN repetidas (REP-PCR. La susceptibilidad antimicrobiana y la producción de BLEE se determinaron de acuerdo con las normas de NCCLS. Las beta-lactamasas se evaluaron por isoelectroenfoque y PCR. El 80% de estos aislamientos se asociaron con infección nosocomial y de éstos, el 91,7% provenía de unidades de cuidado intensivo. La susceptibilidad antibiótica mostró 13 patrones de resistencia; 87% de los aislamientos presentó corresistencia a amikacina, 53% a gentamicina, 33,3% a ciprofloxacina, 40% a cefepime, 66,7% a piperacilina/tazobactam, 60% trimetoprim/sulfametoxazol y 46,7% a cloranfenicol. Todos fueron sensibles a imipenem. En la mayoría de los aislamientos se detectó producción simultánea de beta-lactamasas del tipo TEM y SHV y el 93,3% produjo ceftazidimasa de pI 8.2 del tipo SHV-5. Los 15 aislamientos fueron agrupados por PFGE y REP-PCR en 11 y 12 patrones electroforéticos, respectivamente. Esta variabilidad genética está relacionada con infecciones nosocomiales de origen endógeno más que por infecciones cruzadas.

  4. Evaluación crítica del sistema de diagnóstico de la diabetes mellitus, propuesto por la Asociación Americana de Diabetes Critical evaluation of the diagnostic system of diabetes mellitus proposed by the American Diabetes Association

    Directory of Open Access Journals (Sweden)

    Roberto M. González Suárez

    2002-08-01

    Full Text Available Se realizó un estudio retrospectivo en 370 sujetos con trastornos de tolerancia a la glucosa, para evaluar la metodología de diagnóstico de la diabetes mellitus tipo 2, que prescinde de la prueba de tolerancia a la glucosa oral (PTG, propuesto por la ADA. Se encontró que la glucemia en ayunas permitió diagnosticar el 27 % de los diabéticos y el 56 % de los sujetos con trastornos de la regulación de la glucemia, que se hubieran diagnosticado siguiendo los criterios de la OMS que incluyen la PTG; la tercera parte de los casos clasificados como normales según la glucemia en ayunas solamente, presentó algún tipo de trastorno de la tolerancia a la glucosa. Se comprobó que los diabéticos detectados mediante la hiperglucemia a la segunda hora de la PTG, presentan niveles superiores de glucosa circulante durante la prueba, evaluados por el área total bajo la curva de glucosa. El patrón de respuesta insulínica de estos casos se caracterizó por una respuesta inicial disminuida que se incrementó hasta alcanzar valores máximos a la segunda hora; la frecuencia de casos con baja respuesta insulínica y/o resistencia a la insulina fue alta. Se concluyó que la glucemia en ayunas, como única prueba, no tiene sensibilidad suficiente para detectar trastornos de la tolerancia a la glucosa y diabetes mellitus y los casos con hiperglucemia posprandial únicamente, y que por ello solo pueden ser detectados por la PTG, presentan trastornos metabólicos importantes que requieren su detección y tratamiento precoz por lo cual es recomendable el uso sistemático de la PTG en el diagnóstico de la diabetes mellitus.A retrospective study was carried out in 370 subjects with glucose tolerance disorders aimed at evaluating the methodology for diagnosing type 2 diabetes mellitus that leaves out the oral glucose tolerance test (GTT proposed by the ADA, It was found that fasting glycaemia allowed to diagnose 27 % of the diabetics and 56 % of the individuals

  5. Inter-laboratory analysis of selected genetically modified plant reference materials with digital PCR.

    Science.gov (United States)

    Dobnik, David; Demšar, Tina; Huber, Ingrid; Gerdes, Lars; Broeders, Sylvia; Roosens, Nancy; Debode, Frederic; Berben, Gilbert; Žel, Jana

    2018-01-01

    Digital PCR (dPCR), as a new technology in the field of genetically modified (GM) organism (GMO) testing, enables determination of absolute target copy numbers. The purpose of our study was to test the transferability of methods designed for quantitative PCR (qPCR) to dPCR and to carry out an inter-laboratory comparison of the performance of two different dPCR platforms when determining the absolute GM copy numbers and GM copy number ratio in reference materials certified for GM content in mass fraction. Overall results in terms of measured GM% were within acceptable variation limits for both tested dPCR systems. However, the determined absolute copy numbers for individual genes or events showed higher variability between laboratories in one third of the cases, most possibly due to variability in the technical work, droplet size variability, and analysis of the raw data. GMO quantification with dPCR and qPCR was comparable. As methods originally designed for qPCR performed well in dPCR systems, already validated qPCR assays can most generally be used for dPCR technology with the purpose of GMO detection. Graphical abstract The output of three different PCR-based platforms was assessed in an inter-laboratory comparison.

  6. Genetic variability of Pantaneiro horse using RAPD-PCR markers

    OpenAIRE

    Egito,Andréa Alves do; Fuck,Beatriz Helena; McManus,Concepta; Paiva,Samuel Rezende; Albuquerque,Maria do Socorro Maués; Santos,Sandra Aparecida; Abreu,Urbano Gomes Pinto de; Silva,Joaquim Augusto da; Sereno,Fabiana Tavares Pires de Souza; Mariante,Arthur da Silva

    2007-01-01

    Blood samples were collected from Pantaneiro Horses in five regions of Mato Grosso do Sul and Mato Grosso States. Arabian, Mangalarga Marchador and Thoroughbred were also included to estimate genetic distances and the existing variability among and within these breeds by RAPD-PCR (Random Amplified Polymorphic DNA - Polymerase Chain Reaction) molecular markers. From 146 primers, 13 were chosen for amplification and 44 polymorphic bands were generated. The analysis of molecular variance (AMOVA)...

  7. Multiplex PCR Assay for Identification of Human Diarrheagenic Escherichia coli

    OpenAIRE

    Toma, Claudia; Lu, Yan; Higa, Naomi; Nakasone, Noboru; Chinen, Isabel; Baschkier, Ariela; Rivas, Marta; Iwanaga, Masaaki

    2003-01-01

    A multiplex PCR assay for the identification of human diarrheagenic Escherichia coli was developed. The targets selected for each category were eae for enteropathogenic E. coli, stx for Shiga toxin-producing E. coli, elt and est for enterotoxigenic E. coli, ipaH for enteroinvasive E. coli, and aggR for enteroaggregative E. coli. This assay allowed the categorization of a diarrheagenic E. coli strain in a single reaction tube.

  8. Multiple Hotspot Mutations Scanning by Single Droplet Digital PCR.

    Science.gov (United States)

    Decraene, Charles; Silveira, Amanda B; Bidard, François-Clément; Vallée, Audrey; Michel, Marc; Melaabi, Samia; Vincent-Salomon, Anne; Saliou, Adrien; Houy, Alexandre; Milder, Maud; Lantz, Olivier; Ychou, Marc; Denis, Marc G; Pierga, Jean-Yves; Stern, Marc-Henri; Proudhon, Charlotte

    2018-02-01

    Progress in the liquid biopsy field, combined with the development of droplet digital PCR (ddPCR), has enabled noninvasive monitoring of mutations with high detection accuracy. However, current assays detect a restricted number of mutations per reaction. ddPCR is a recognized method for detecting alterations previously characterized in tumor tissues, but its use as a discovery tool when the mutation is unknown a priori remains limited. We established 2 ddPCR assays detecting all genomic alterations within KRAS exon 2 and EGFR exon 19 mutation hotspots, which are of clinical importance in colorectal and lung cancer, with use of a unique pair of TaqMan ® oligoprobes. The KRAS assay scanned for the 7 most common mutations in codons 12/13 but also all other mutations found in that region. The EGFR assay screened for all in-frame deletions of exon 19, which are frequent EGFR-activating events. The KRAS and EGFR assays were highly specific and both reached a limit of detection of <0.1% in mutant allele frequency. We further validated their performance on multiple plasma and formalin-fixed and paraffin-embedded tumor samples harboring a panel of different KRAS or EGFR mutations. This method presents the advantage of detecting a higher number of mutations with single-reaction ddPCRs while consuming a minimum of patient sample. This is particularly useful in the context of liquid biopsy because the amount of circulating tumor DNA is often low. This method should be useful as a discovery tool when the tumor tissue is unavailable or to monitor disease during therapy. © 2017 American Association for Clinical Chemistry.

  9. an overview on the application of polymerase chain reaction (pcr)

    African Journals Online (AJOL)

    DR. AMINU

    Hill New York Pp818. Chul, W.P., Jang-Hee, H., Jin-Hyeok, J. et al (2004). Detection rates of Bacteria in chronic Otitis. Media with Effusion in Children, Journal of. Korean Medical Sciences 19: 735-738. Cockerill, F.R. and Smith, T.F. (2002). Rapid-Cycle real time PCR: A revolution of clinical. Microbiology. ASM News 68:2.

  10. Validation of kinetics similarity in qPCR

    Czech Academy of Sciences Publication Activity Database

    Bar, T.; Kubista, Mikael; Tichopád, Aleš

    2012-01-01

    Roč. 40, č. 4 (2012), s. 1395-1406 ISSN 0305-1048 R&D Projects: GA ČR GAP303/10/1338; GA ČR GA301/09/1752 Institutional research plan: CEZ:AV0Z50520701 Keywords : REAL-TIME PCR * POLYMERASE-CHAIN-REACTION * SYBR-GREEN-I Subject RIV: EB - Genetics ; Molecular Biology Impact factor: 8.278, year: 2012

  11. Mycobacterium bovis in milk samples: a preliminary investigation using PCR

    International Nuclear Information System (INIS)

    Achel, D.G.; Gyamfi, O.K.; Broni, F.; Gomda, Y.; Brown, C.A.

    2007-01-01

    PCR was used to screen milk samples (n=41) for Mycobacterium bovis. DNA samples were obtained through concentration by 50% sucrose addition and centrifugation. Sixteen (16) samples (or 39%) were positive for M. Bovis DNA and the rest 25 (or 61%) were negative. All four kraals had some samples testing positive for M. bovis; the highest being 50% (5/10) and the lowest being 13% (2/15). (au)

  12. Extraction of PCR-amplifiable genomic DNA from Bacillus anthracisspores

    Energy Technology Data Exchange (ETDEWEB)

    Torok, Tamas

    2003-05-19

    Bacterial endospore disruption and nucleic acid extractionresulting in DNA of PCR-amplifiable quality and quantity are not trivial.Responding to the needs of the Hazardous Materials Response Unit (HMRU),Laboratory Division, Federal Bureau of Investigation, protocols weredeveloped to close these gaps. Effectiveness and reproducibility of thetechniques were validated with laboratory grown pure spores of Bacillusanthracis and its close phylogenetic neighbors, and with spiked soils anddamaged samples.

  13. Polymerase Chain Reaction (Pcr) Assay to Detect Hepatitis C Virus

    International Nuclear Information System (INIS)

    Lina MR; Dadang S; Budiman Bela

    2004-01-01

    Research on the detection of hepatitis C virus in blood serum using PCR technique has been carried out. Amount of 50 blood serum from laboratory of Indonesia Red Cross (Palang Merah Indonesia = PMI) and RSCM hospital as samples, were used in this research. Lysis of virus cell and extraction of RNA virus as a preliminary treatment of the sample, was done with BOOM method using guanidine thiocyanate and diatomaceous earth, respectively. Synthesis of cDNA from RNA as an extraction product mentioned above, was carried out by means of reverse-transcriptase and RNA-se inhibitor. Amplification of cDNA was done with nested PCR technique that was performed with two times PCR processes using two pairs of oligonucleotide primers for each process. The amplified DNA was detected by agarose gel electrophoresis and ethidium bromide staining. Subsequently, the DNA was visualized with UV transilluminator. Result shows that of 50 blood serum samples, 13 serum were positive for RNA HCV that were performed with the present of specific DNA band on agarose gel. (author)

  14. SCREENING OF COMMON FLAX FAD GENES BY PCR

    Directory of Open Access Journals (Sweden)

    Veronika Štefúnová

    2013-02-01

    Full Text Available Currently, flax (Linum usitatissimum L. is an important crop from commercial and economical aspects. In the spotlight is the linseed oil as a source of α-linolenic acid. The aim of presented study was to analyse fatty acid desaturase (FAD genes in flax. Several genotypes of flax (Hohenheim, La Plata 1938, Redwing USA and Escalina were used. The primers described by Vrinten et al. (2005 were used for PCR amplification reactions. Two FAD3 genes, LuFAD3A and LuFAD3B, were identified in a genome of flax. Subsequently the nucleotide sequences between origins and genotypes of flax FAD genes were compared. Primarily were used the nucleotide sequences of FAD2 and FAD3C genes available in NCBI database. Differences were found using BLAST program in nucleotide sequences of FAD genes and the specific primers were designed to amplify a specific target sequences in a genome of flax. These primers were used in PCR amplification reactions to identification of FAD2 and FAD3C genes. The PCR products were separated by electrophoresis on agarose gel.

  15. Rapid diagnosis of aneuploidy using segmental duplication quantitative fluorescent PCR.

    Directory of Open Access Journals (Sweden)

    Xiangdong Kong

    Full Text Available The aim of this study was use a simple and rapid procedure, called segmental duplication quantitative fluorescent polymerase chain reaction (SD-QF-PCR, for the prenatal diagnosis of fetal chromosomal aneuploidies. This method is based on the co-amplification of segmental duplications located on two different chromosomes using a single pair of fluorescent primers. The PCR products of different sizes were subsequently analyzed through capillary electrophoresis, and the aneuploidies were determined based on the relative dosage between the two chromosomes. Each primer set, containing five pairs of primers, was designed to simultaneously detect aneuploidies located on chromosomes 21, 18, 13, X and Y in a single reaction. We applied these two primer sets to DNA samples isolated from individuals with trisomy 21 (n = 36; trisomy 18 (n = 6; trisomy 13 (n = 4; 45, X (n = 5; 47, XXX (n = 3; 48, XXYY (n = 2; and unaffected controls (n = 40. We evaluated the performance of this method using the karyotyping results. A correct and unambiguous diagnosis with 100% sensitivity and 100% specificity, was achieved for clinical samples examined. Thus, the present study demonstrates that SD-QF-PCR is a robust, rapid and sensitive method for the diagnosis of common aneuploidies, and these analyses can be performed in less than 4 hours for a single sample, providing a competitive alternative for routine use.

  16. Authentication of Meat Species in Sucuk by Multiplex PCR

    Directory of Open Access Journals (Sweden)

    Osman İrfan İLHAK

    2015-01-01

    Full Text Available The identification of meat species used in meat products is important by reason of economic considerations, religious factors, verification of label, and prevention of unfair-market competition. In this paper, multiplex PCR method was experienced for routine detection of equine (horse and donkey, poultry (chicken and turkey, pig and cattle meat in sucuk (sausage. The primers used for these animals generated specific fragments, and they did not show cross reactions with the DNA from the other genus of animal. After multiplex PCR was successfully optimized, a field study was carried out to investigate the presence of horse, donkey, chicken, turkey and pig meat in 50 sucuks (30 beef and 20 beef + poultry collected from markets. The result of the field study indicated that 23.3% of 30 beef sucuk samples were containing poultry meat. None of the 50 sucuk samples was containing pig meat, but one (2% of the samples generated equine fragment. The present study showed that the multiplex PCR method can be used for routine analysis of meat species identification, verification and control of label information of meat products.

  17. Direct-to-PCR tissue preservation for DNA profiling.

    Science.gov (United States)

    Sorensen, Amy; Berry, Clare; Bruce, David; Gahan, Michelle Elizabeth; Hughes-Stamm, Sheree; McNevin, Dennis

    2016-05-01

    Disaster victim identification (DVI) often occurs in remote locations with extremes of temperatures and humidities. Access to mortuary facilities and refrigeration are not always available. An effective and robust DNA sampling and preservation procedure would increase the probability of successful DNA profiling and allow faster repatriation of bodies and body parts. If the act of tissue preservation also released DNA into solution, ready for polymerase chain reaction (PCR), the DVI process could be further streamlined. In this study, we explored the possibility of obtaining DNA profiles without DNA extraction, by adding aliquots of preservative solutions surrounding fresh human muscle and decomposing human muscle and skin tissue samples directly to PCR. The preservatives consisted of two custom preparations and two proprietary solutions. The custom preparations were a salt-saturated solution of dimethyl sulfoxide (DMSO) with ethylenediaminetetraacetic (EDTA) and TENT buffer (Tris, EDTA, NaCl, Tween 20). The proprietary preservatives were DNAgard (Biomatrica(®)) and Tissue Stabilising Kit (DNA Genotek). We obtained full PowerPlex(®) 21 (Promega) and GlobalFiler(®) (Life Technologies) DNA profiles from fresh and decomposed tissue preserved at 35 °C for up to 28 days for all four preservatives. The preservative aliquots removed from the fresh muscle tissue samples had been stored at -80 °C for 4 years, indicating that long-term archival does not diminish the probability of successful DNA typing. Rather, storage at -80 °C seems to reduce PCR inhibition.

  18. Aspergillus section Fumigati typing by PCR-restriction fragment polymorphism.

    Science.gov (United States)

    Staab, Janet F; Balajee, S Arunmozhi; Marr, Kieren A

    2009-07-01

    Recent studies have shown that there are multiple clinically important members of the Aspergillus section Fumigati that are difficult to distinguish on the basis of morphological features (e.g., Aspergillus fumigatus, A. lentulus, and Neosartorya udagawae). Identification of these organisms may be clinically important, as some species vary in their susceptibilities to antifungal agents. In a prior study, we utilized multilocus sequence typing to describe A. lentulus as a species distinct from A. fumigatus. The sequence data show that the gene encoding beta-tubulin, benA, has high interspecies variability at intronic regions but is conserved among isolates of the same species. These data were used to develop a PCR-restriction fragment length polymorphism (PCR-RFLP) method that rapidly and accurately distinguishes A. fumigatus, A. lentulus, and N. udagawae, three major species within the section Fumigati that have previously been implicated in disease. Digestion of the benA amplicon with BccI generated unique banding patterns; the results were validated by screening a collection of clinical strains and by in silico analysis of the benA sequences of Aspergillus spp. deposited in the GenBank database. PCR-RFLP of benA is a simple method for the identification of clinically important, similar morphotypes of Aspergillus spp. within the section Fumigati.

  19. Aspergillus Section Fumigati Typing by PCR-Restriction Fragment Polymorphism▿

    Science.gov (United States)

    Staab, Janet F.; Balajee, S. Arunmozhi; Marr, Kieren A.

    2009-01-01

    Recent studies have shown that there are multiple clinically important members of the Aspergillus section Fumigati that are difficult to distinguish on the basis of morphological features (e.g., Aspergillus fumigatus, A. lentulus, and Neosartorya udagawae). Identification of these organisms may be clinically important, as some species vary in their susceptibilities to antifungal agents. In a prior study, we utilized multilocus sequence typing to describe A. lentulus as a species distinct from A. fumigatus. The sequence data show that the gene encoding β-tubulin, benA, has high interspecies variability at intronic regions but is conserved among isolates of the same species. These data were used to develop a PCR-restriction fragment length polymorphism (PCR-RFLP) method that rapidly and accurately distinguishes A. fumigatus, A. lentulus, and N. udagawae, three major species within the section Fumigati that have previously been implicated in disease. Digestion of the benA amplicon with BccI generated unique banding patterns; the results were validated by screening a collection of clinical strains and by in silico analysis of the benA sequences of Aspergillus spp. deposited in the GenBank database. PCR-RFLP of benA is a simple method for the identification of clinically important, similar morphotypes of Aspergillus spp. within the section Fumigati. PMID:19403766

  20. Validation of chimerism in pediatric recipients of allogeneic hematopoietic stem cell transplantation (HSCT) a comparison between two methods: real-time PCR (qPCR) vs. variable number tandem repeats PCR (VNTR PCR).

    Science.gov (United States)

    Kletzel, Morris; Huang, Wei; Olszewski, Marie; Khan, Sana

    2013-01-01

    Post-hematopoietic stem cell transplantation (HSCT) chimerism monitoring is important to assess relapse and therapeutic intervention. The purpose of our study is to compare two methods variable number tandem repeats (VNTR) vs. quantitative real- time polymerase chain reaction (qPCR) in terms of determining chimerism. 127 (peripheral blood n=112, bone marrow n=15) samples were simultaneously tested by VNTR using APO-B, D1S80, D1S111, D17S30, gene loci SRY and ZP3 and qPCR using 34 assays (CA001-CA034) that are designed to a bi-allelic insertion/deletion (indel) polymorphism in the human genome. Samples were separated in three subsets: total WBC, T-cell and Myeloid cells. Extraction of DNA was performed then quantified. We analyzed column statistics, paired t-test and regression analysis for both methods. There was complete correlation between the two methods. The simplicity and rapidity of the test results from the qPCR method is more efficient and accurate to assess chimerism.

  1. Diagnosis of aerobic vaginitis by quantitative real-time PCR.

    Science.gov (United States)

    Rumyantseva, T A; Bellen, G; Savochkina, Y A; Guschin, A E; Donders, G G G

    2016-07-01

    To evaluate a real-time PCR-based technique to quantify bacteria associated with aerobic vaginitis (AV) as a potential test. Vaginal samples from 100 women were tested by wet-mount microscopy, gram stain and quantitative real-time PCR targeting Enterobacteriacea, Staphylococcus spp., Streptococcus spp., Enterococcus spp., Escherichia coli, Streptococcus agalactiae, S. aureus; Lactobacillus spp. AV diagnosis obtained by wet-mount microscopy was used as reference. Some level of AV was diagnosed in 23 (23.7 %) cases. Various concentrations of Enterobacteriacea, Staphylococcus spp., Streptococcus spp. were detected an all patients. Enterococcus spp. were detected in 76 (78.3 %) cases. Summarized concentrations of aerobes were tenfold higher in AV-positive compared to AV-negative cases [7.30lg vs 6.06lg (p = 0.02)]. Concentrations of aerobes in severe, moderate and light AV cases did not vary significantly (p = 0.14). Concentration of lactobacilli was 1000-fold lower in AV-positive cases compared to normal cases (5.3lg vs 8.3lg, p AV-positive cases [19/22 (86.4 %) samples]. The relation of high loads of aerobes to the low numbers of Lactobacilli are a reliable marker for the presence of AV and could substitute microscopy as a test. PCR may be a good standardized substitution for AV diagnosis in settings where well-trained microscopists are lacking.

  2. Using PCR to Target Misconceptions about Gene Expression

    Directory of Open Access Journals (Sweden)

    Leslie K. Wright

    2013-02-01

    Full Text Available We present a PCR-based laboratory exercise that can be used with first- or second-year biology students to help overcome common misconceptions about gene expression. Biology students typically do not have a clear understanding of the difference between genes (DNA and gene expression (mRNA/protein and often believe that genes exist in an organism or cell only when they are expressed. This laboratory exercise allows students to carry out a PCR-based experiment designed to challenge their misunderstanding of the difference between genes and gene expression. Students first transform E. coli with an inducible GFP gene containing plasmid and observe induced and un-induced colonies. The following exercise creates cognitive dissonance when actual PCR results contradict their initial (incorrect predictions of the presence of the GFP gene in transformed cells. Field testing of this laboratory exercise resulted in learning gains on both knowledge and application questions on concepts related to genes and gene expression.

  3. Use of a Mycoplasma suis-PCR protocol for screening a population of captive peccaries (Tayassu tajacu and Tayassu pecari Uso de um protocolo de PCR para a detecção de Mycoplasma suis para avaliação de uma população de catetos e queixadas de cativeiro (Tayassu tajacu and Tayassu pecari

    Directory of Open Access Journals (Sweden)

    Rafael Felipe da Costa Vieira

    2011-03-01

    Full Text Available Mycoplasma suis is a hemotropic bacteria of red blood cells and the causative agent of swine eperythrozoonosis. Diagnosis of infection may be reached by direct examination of blood smears; however, the use of polymerase chain reaction (PCR of the 16S RNA gene of M. suis improves the sensitivity and specificity of detection. The aim of this study was to screen peccaries (Tayassu tajacu and T. pecari for M. suis infection using a specific conventional PCR. A total of 28 blood samples from captive collared and white-lipped peccaries were collected, DNA extracted and a specific M. suis PCR assay performed. All samples were negatives by both blood smear examination and PCR testing. To verify the presence of amplifiable DNA, PCR for beta-actin gene was performed in all samples. This study was part of an active surveillance program, which is crucial for monitoring animal health status, particularly in wildlife species.Mycoplasma suis é uma bactéria hemotrópica dos eritrócitos e é o agente causador da eperitrozoonose suína. O diagnóstico da infecção pode ser realizado pelo exame direto de esfregaços sanguíneos; entretanto, o uso da reação em cadeia da polimerase (PCR baseada no gene 16S RNA de M. suis aumenta a sensibilidade e especificidade da detecção. O objetivo deste estudo foi avaliar catetos e queixadas (Tayassu tajacu e T. pecari para a infecção por M. suis, utilizando PCR convencional específico. Um total de 28 amostras de sangue de catetos e queixadas de cativeiro foram coletadas, o DNA foi extraído e a PCR específica para a detecção de M. suis realizada. Todas as amostras foram negativas pelo esfregaço sanguíneo e PCR. Para verificar a presença de DNA amplificável, PCR para o gene da beta actina foi realizada em todas as amostras. Este estudo foi parte de um programa de vigilância ativa, o qual é crucial para o monitoramento do estado de saúde animal, particularmente em espécies selvagens.

  4. Purification of nanoparticle PCR products and their topography observed with AFM

    International Nuclear Information System (INIS)

    Mi Lijuan; Wang Hubin; Chinese Academy of Sciences, Beijing; Li Bin; Zhou Hualan; Hu Jun

    2007-01-01

    Nanoparticle PCR (NP-PCR) is a new method to optimize PCR amplification. Suitable amount of Au nanoparticles can improve specificity, sensitivity and extension rate of PCR. In this paper, we compare efficiency of purifying NP-PCR products with different methods. In addition, topographies of DNA products in NP-PCR were observed with atomic force microscope (AFM). The results show that most of DNA products purified directly by routing method remain almost free due to less effect of nanoparticales. The yields decrease when the AuNPs were removed by high-speed centrifugation. A little amount of DNA subsided with AuNPs. (authors)

  5. A new detection method for the K variant of butyrylcholinesterase based on PCR primer introduced restriction analysis (PCR-PIRA).

    Science.gov (United States)

    Shibuta, K; Abe, M; Suzuki, T

    1994-01-01

    The K variant of human butyrylcholinesterase is caused by a G/A transition in the butyrylcholinesterase gene, which neither creates nor destroys any restriction site. In an attempt to detect the K variant both simply and rapidly, we developed a two step method of "PCR primer introduced restriction analysis" (PCR-PIRA). The first step was used to introduce a new Fun4HI site into the normal allele for a screening test, while the second step was performed to create a new MaeIII site on the variant allele for a specific test. This method thus enabled us to distinguish clearly the K variant from the normal allele, and also showed that the frequency of the K variant allele is 0.164 in the Japanese population. Images PMID:7966197

  6. Implementation of polymerase chain reaction (PCR and Real-Time PCR in quick identification of bovine herpesvirus 1

    Directory of Open Access Journals (Sweden)

    Milić Nenad

    2010-01-01

    Full Text Available Examinations were performed on 65 samples of nasal smeas taken from calves and young cows with clinical symptoms of respiratory infection to determine the presence of the bovine herpes virus 1 using parallel implementation of molecular and standard methods of virological diagnostics. The appearance of a cytopathogenic effect (CPE was not established in inoculated cell lines 24h, 48h and 72h following inoculation, or after two successive passages of the examined material sample through these cell lines. The application of polymerize chain reaction (PCR using a primer for glucoprotein B and thymidine - kinasis, established the presence of bovine herpes virus 1 nucleic acid in one sample of a bovine nasal smear, while the presence of this virus was established in three samples in an examination of the nasal smear samples using the Real-Time PCR method.

  7. A comparison of QuantStudio™ 3D Digital PCR and ARMS-PCR for measuring plasma EGFR T790M mutations of NSCLC patients.

    Science.gov (United States)

    Feng, Qin; Gai, Fei; Sang, Yaxiong; Zhang, Jie; Wang, Ping; Wang, Yue; Liu, Bing; Lin, Dongmei; Yu, Yang; Fang, Jian

    2018-01-01

    The AURA3 clinical trial has shown that advanced non-small cell lung cancer (NSCLC) patients with EGFR T790M mutations in circulating tumor DNA (ctDNA) could benefit from osimertinib. The aim of this study was to assess the usefulness of QuantStudio™ 3D Digital PCR System platform for the detection of plasma EGFR T790M mutations in NSCLC patients, and compare the performances of 3D Digital PCR and ARMS-PCR. A total of 119 Chinese patients were enrolled in this study. Mutant allele frequency of plasma EGFR T790M was detected by 3D Digital PCR, then 25 selected samples were verified by ARMS-PCR and four of them were verified by next generation sequencing (NGS). In total, 52.94% (69/119) had EGFR T790M mutations detected by 3D Digital PCR. In 69 positive samples, the median mutant allele frequency (AF) was 1.09% and three cases presented low concentration (AF Digital PCR) was identified as T790M- by ARMS-PCR. Four samples were identified as T790M+ by both NGS and 3D Digital PCR, and typically three samples (3/4) presented at a low ratio (AF Digital PCR is a novel method with high sensitivity and specificity to detect EGFR T790M mutation in plasma.

  8. Development of Nested PCR, Multiplex PCR, and Loop-Mediated Isothermal Amplification Assays for Rapid Detection of Cylindrocladium scoparium on Eucalyptus

    Directory of Open Access Journals (Sweden)

    Tian-Min Qiao

    2016-10-01

    Full Text Available Eucalyptus dieback disease, caused by Cylindrocladium scoparium, has occurred in last few years in large Eucalyptus planting areas in China and other countries. Rapid, simple, and reliable diagnostic techniques are desired for the early detection of Eucalyptus dieback of C. scoparium prior to formulation of efficient control plan. For this purpose, three PCR-based methods of nested PCR, multiplex PCR, loop-mediated isothermal amplification (LAMP were developed for detection of C. scoparium based on factor 1-alpha (tef1 and beta-tubulin gene in this study. All of the three methods showed highly specific to C. scoparium. The sensitivities of the nested PCR and LAMP were much higher than the multiplex PCR. The sensitivity of multiplex PCR was also higher than regular PCR. C. scoparium could be detected within 60 min from infected Eucalyptus plants by LAMP, while at least 2 h was needed by the rest two methods. Using different Eucalyptus tissues as samples for C. scoparium detection, all of the three PCR-based methods showed much better detection results than regular PCR. Base on the results from this study, we concluded that any of the three PCR-based methods could be used as diagnostic technology for the development of efficient strategies of Eucalyptus dieback disease control. Particularly, LAMP was the most practical method in field application because of its one-step and rapid reaction, simple operation, single-tube utilization, and simple visualization of amplification products.

  9. Development of Nested PCR, Multiplex PCR, and Loop-Mediated Isothermal Amplification Assays for Rapid Detection of Cylindrocladium scoparium on Eucalyptus

    Science.gov (United States)

    Qiao, Tian-Min; Zhang, Jing; Li, Shu-Jiang; Han, Shan; Zhu, Tian-Hui

    2016-01-01

    Eucalyptus dieback disease, caused by Cylindrocladium scoparium, has occurred in last few years in large Eucalyptus planting areas in China and other countries. Rapid, simple, and reliable diagnostic techniques are desired for the early detection of Eucalyptus dieback of C. scoparium prior to formulation of efficient control plan. For this purpose, three PCR-based methods of nested PCR, multiplex PCR, loop-mediated isothermal amplification (LAMP) were developed for detection of C. scoparium based on factor 1-alpha (tef1) and beta-tubulin gene in this study. All of the three methods showed highly specific to C. scoparium. The sensitivities of the nested PCR and LAMP were much higher than the multiplex PCR. The sensitivity of multiplex PCR was also higher than regular PCR. C. scoparium could be detected within 60 min from infected Eucalyptus plants by LAMP, while at least 2 h was needed by the rest two methods. Using different Eucalyptus tissues as samples for C. scoparium detection, all of the three PCR-based methods showed much better detection results than regular PCR. Base on the results from this study, we concluded that any of the three PCR-based methods could be used as diagnostic technology for the development of efficient strategies of Eucalyptus dieback disease control. Particularly, LAMP was the most practical method in field application because of its one-step and rapid reaction, simple operation, single-tube utilization, and simple visualization of amplification products. PMID:27721691

  10. Development of Nested PCR, Multiplex PCR, and Loop-Mediated Isothermal Amplification Assays for Rapid Detection of Cylindrocladium scoparium on Eucalyptus.

    Science.gov (United States)

    Qiao, Tian-Min; Zhang, Jing; Li, Shu-Jiang; Han, Shan; Zhu, Tian-Hui

    2016-10-01

    Eucalyptus dieback disease, caused by Cylindrocladium scoparium , has occurred in last few years in large Eucalyptus planting areas in China and other countries. Rapid, simple, and reliable diagnostic techniques are desired for the early detection of Eucalyptus dieback of C. scoparium prior to formulation of efficient control plan. For this purpose, three PCR-based methods of nested PCR, multiplex PCR, loop-mediated isothermal amplification (LAMP) were developed for detection of C. scoparium based on factor 1-alpha (tef1) and beta-tubulin gene in this study. All of the three methods showed highly specific to C. scoparium . The sensitivities of the nested PCR and LAMP were much higher than the multiplex PCR. The sensitivity of multiplex PCR was also higher than regular PCR. C. scoparium could be detected within 60 min from infected Eucalyptus plants by LAMP, while at least 2 h was needed by the rest two methods. Using different Eucalyptus tissues as samples for C. scoparium detection, all of the three PCR-based methods showed much better detection results than regular PCR. Base on the results from this study, we concluded that any of the three PCR-based methods could be used as diagnostic technology for the development of efficient strategies of Eucalyptus dieback disease control. Particularly, LAMP was the most practical method in field application because of its one-step and rapid reaction, simple operation, single-tube utilization, and simple visualization of amplification products.

  11. OPTIMIZACIÓN DE UN PROTOCOLO DEL AISLAMIENTO DEL ADN Y DE UN SISTEMA DE AMPLIFICACIÓN ISSR-PCR PARA Ceratozamia mexicana Brongn. (Zamiaceae.

    Directory of Open Access Journals (Sweden)

    Nadia Guadalupe Sánchez-Coello

    2012-01-01

    Full Text Available La mayoría de las cícadas contienen altas concentraciones de aceites esenciales, flavonoides, polifenoles y polisacáridos que interfieren en la extracción de ADN, causando productos de amplificación errados o inhibiendo la PCR. La optimización del aislamiento del ADN y el empleo de iniciadores de secuencias intergénicas repetidas simples (ISSRs se investigaron en Ceratozamia mexicana Brongn., una cícada mexicana en peligro de extinción. El ADN obtenido de tejido foliar fresco, con un amortiguador modificado de cetil trimetil amonio, nos permitió obtener un ADN de buena calidad, sin pigmentos coloridos o contaminantes. La modificación al protocolo de extracción de ADN, basado en CTAB, fue un prelavado por 1 h, del tejido foliar, con una solución de 0.7 M de NaCl, para facilitar la lisis celular. El ADN extraído exitosamente se amplificó por PCR, usando seis iniciadores arbitrarios ISSR. Se observaron productos de amplificación reproducibles en todas las reacciones de PCR. Nuestros resultados muestran que la implementación mejora significativamente la calidad del ADN obtenido, usando una concentración baja de iniciadores (25 pM. Se detectaron 23 bandas fuertes, nueve de las cuales fueron polimórficas. Los resultados indican que el protocolo de optimización del aislamiento del ADN y en el sistema de PCR es viable para futuros trabajos en esta especie. Este trabajo es el primer protocolo de extracción de ADN y de ISSR reportado para esta especie ornamental en peligro de extinción.

  12. Revealing the microbiota of marketed edible insects through PCR-DGGE, metagenomic sequencing and real-time PCR.

    Science.gov (United States)

    Osimani, Andrea; Milanović, Vesna; Garofalo, Cristiana; Cardinali, Federica; Roncolini, Andrea; Sabbatini, Riccardo; De Filippis, Francesca; Ercolini, Danilo; Gabucci, Claudia; Petruzzelli, Annalisa; Tonucci, Franco; Clementi, Francesca; Aquilanti, Lucia

    2018-07-02

    The present study aimed to identify the microbiota present in six species of processed edible insects produced in Thailand and marketed worldwide via the internet, namely, giant water bugs (Belostoma lutarium), black ants (Polyrhachis), winged termites (alates, Termitoidae), rhino beetles (Hyboschema contractum), mole crickets (Gryllotalpidae), and silkworm pupae (Bombyx mori). For each species, two samples of boiled, dried and salted insects were purchased. The microbial DNA was extracted from the insect samples and subjected to polymerase chain reaction-denaturing gradient gel electrophoresis (PCR-DGGE), high-throughput sequencing and qualitative real-time PCR assays. The microbiota of the analyzed samples were widely characterized by the presence of spore-forming bacteria mainly represented by the genera Bacillus and Clostridium. Moreover, the genera Anaerobacillus, Paenibacillus, Geobacillus, Pseudomonas, Stenotrophomonas, Massilia, Delftia, Lactobacillus, Staphylococcus, Streptococcus, Vagococcus, and Vibrio were also detected. Real-time PCR allowed for ascertainment of the absence of Coxiella burnetii, Shiga toxin-producing E. coli (STEC), and Pseudomonas aeruginosa in all samples. The results of this study confirm the importance of combining different molecular techniques to characterize the biodiversity of complex ecosystems such as edible insects. The presence of potential human pathogens suggests the need for a careful application of good manufacturing practices during insect processing. This study provides further data that will be useful in risk analyses of edible insects as a novel food source. Copyright © 2018 Elsevier B.V. All rights reserved.

  13. Highly efficient PCR assay to discriminate allelic DNA methylation status using whole genome amplification

    Directory of Open Access Journals (Sweden)

    Ito Takashi

    2011-06-01

    Full Text Available Abstract Background We previously developed a simple method termed HpaII-McrBC PCR (HM-PCR to discriminate allelic methylation status of the genomic sites of interest, and successfully applied it to a comprehensive analysis of CpG islands (CGIs on human chromosome 21q. However, HM-PCR requires 200 ng of genomic DNA to examine one target site, thereby precluding its application to such samples that are limited in quantity. Findings We developed HpaII-McrBC whole-genome-amplification PCR (HM-WGA-PCR that uses whole-genome-amplified DNA as the template. HM-WGA-PCR uses only 1/100th the genomic template material required for HM-PCR. Indeed, we successfully analyzed 147 CGIs by HM-WGA-PCR using only ~300 ng of DNA, whereas previous HM-PCR study had required ~30 μg. Furthermore, we confirmed that allelic methylation status revealed by HM-WGA-PCR is identical to that by HM-PCR in every case of the 147 CGIs tested, proving high consistency between the two methods. Conclusions HM-WGA-PCR would serve as a reliable alternative to HM-PCR in the analysis of allelic methylation status when the quantity of DNA available is limited.

  14. [Sensitivity and specificity of nested PCR pyrosequencing in hepatitis B virus drug resistance gene testing].

    Science.gov (United States)

    Sun, Shumei; Zhou, Hao; Zhou, Bin; Hu, Ziyou; Hou, Jinlin; Sun, Jian

    2012-05-01

    To evaluate the sensitivity and specificity of nested PCR combined with pyrosequencing in the detection of HBV drug-resistance gene. RtM204I (ATT) mutant and rtM204 (ATG) nonmutant plasmids mixed at different ratios were detected for mutations using nested-PCR combined with pyrosequencing, and the results were compared with those by conventional PCR pyrosequencing to analyze the linearity and consistency of the two methods. Clinical specimens with different viral loads were examined for drug-resistant mutations using nested PCR pyrosequencing and nested PCR combined with dideoxy sequencing (Sanger) for comparison of the detection sensitivity and specificity. The fitting curves demonstrated good linearity of both conventional PCR pyrosequencing and nested PCR pyrosequencing (R(2)>0.99, PNested PCR showed a better consistency with the predicted value than conventional PCR, and was superior to conventional PCR for detection of samples containing 90% mutant plasmid. In the detection of clinical specimens, Sanger sequencing had a significantly lower sensitivity than nested PCR pyrosequencing (92% vs 100%, Pnested PCR and Sanger sequencing method, nested PCR pyrosequencing has a higher sensitivity especially in clinical specimens with low viral copies, which can be important for early detection of HBV mutant strains and hence more effective clinical management.

  15. Evolución de la prevalencia de infección por virus de la inmunodeficiencia humana en población reclusa al ingreso en prisión durante el período 1991-1995

    Directory of Open Access Journals (Sweden)

    Martín Vicente

    1997-01-01

    Full Text Available FUNDAMENTO: Las prisiones españolas albergan un elevado número de usuarios de drogas por vía parenteral (UDVP, y otras personas con prácticas de riesgo para la infección por virus de la inmunodeficiencia humana (VIH. El objetivo de este trabajo es conocer la evolución de la prevalencia de infección por VIH en el momento del ingreso en prisión y los factores asociados a la misma en este colectivo, lo que puede permitir una aproximación a la efectividad de las estrategias de reducción de riesgos y contribuir a mejorarlas. MÉTODOS: Todas aquellas personas que ingresaron en un centro penitenciario provincial del noroeste español entre los años 1991 y 1995. Se recogieron variables socio-demográficas, penitenciarias y factores de riesgo para VIH. Previo consentimiento, se practicó prueba de infección por VIH (ELISA y Western-blot. RESULTADOS: El 19,4% de los 1663 individuos estudiados estaban infectados por VIH. Destacaban, con significación estadística, las prevalencias en: mujeres (26,0%, el grupo de edad desde 25 a 34 años (29,1%, blancos (20,9%, solteros (22,8%, tatuados (29,9%, los que presentaban antecedentes de autolesiones (42,4%, los UDVP (46,3%, los que reconocieron compartir jeringuillas (61,5 % y aquellos con antecedentes de haber permanecido en prisión un año o más (37,3%. El análisis de regresión logística mostró como predictores de infección VIH: los UDVP, los que ingresaron en 1992, las mujeres, los grupos de edad de 25-34 y de 35-44 años, los tatuados, los que presentaban antecedentes de autolesiones y aquellos con antecedentes de estancia en prisión superior a un año. La etnia gitana presentó menor probabilidad de infección por VIH. La tendencia temporal de la infección VIH estratificada según el antecedente de estancia previa en prisión, mostró un descenso casi significativo (P=0,064. La tendencia de la infección según UDVP no mostró modificación (P=0,16. CONCLUSIONES: Se ha detectado una

  16. Frecuencia de la colonización por Pneumocystis jirovecii en pacientes con Enfermedad Pulmonar Obstructiva Crónica en Bogotá, Colombia

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    Juan Felipe Burbano-Gutiérrez

    2017-04-01

    Full Text Available Antecedentes: la colonización por Pneumocystis jirovecci (P. jirovecii se ha postulado como causa de deterioro de la función pulmonar en pacientes con Enfermedad Pulmonar Obstructiva Crónica (EPOC. Se desconocía la frecuencia de aparición de la colonización por P. jirovencii en esa población en Colombia. Objetivo: documentar la frecuencia de colonización por P. jirovecii en mayores de 40 años con EPOC excluyendo a los pacientes que requirieran manejo inmunosupresor y a las personas infectadas por el Virus de la Inmunodeficiencia Humana (VIH. Materiales y métodos: se trató de un estudio de corte transversal, que contó con muestreo no probabilístico por conveniencia y selección continua de pacientes. Se realizó PCR (reacción en cadena de polimerasa en tiempo real (rt-PCR del esputo inducido con el Kit LighMix de P. jirovecii (Roche®-Suiza amplificándose un fragmento de 244 pares de bases a partir del gen de la glicoproteína de superficie del hongo. Resultados: para una muestra de 52 pacientes en total, se documentó una frecuencia de colonización del 15,4% en todos los participantes mayores de 65 años, quienes además presentaron altos índices de sintomatología según la escala modificada Medical Research Council (MR Cm y el cuestionario de evaluación de la EPOC (CAT. La mayoría de pacientes analizados se clasificó como GOLD D (63% en la clasificación por la Iniciativa Global para la EPOC. Conclusiones: la frecuencia de colonización por P. jirovecii en pacientes con EPOC detectada por rt-PCR en el esputo inducido fue del 15,4%. Este constituye el primer estudio colombiano que evalúa la frecuencia de colonización del hongo.

  17. Autoagresión por quemaduras

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    Iris Montes de Oca

    1996-04-01

    Full Text Available Se realizó un estudio epidemiológico transversal en los casos de autoagresión por quemaduras, que acudieron al Cuerpo de Guardia del Servicio de Quemados del Hospital "Dr. Ernesto Guevara de la Serna", durante el período comprendido entre los meses de febrero de 1993 y febrero de 1994, con la finalidad de determinar el comportamiento de algunas variables de interés en estos casos. El universo de estudio lo constituyeron 57 casos, cuya información se recolectó de la gráfica general del paciente quemado (Modelo 71-05 y de los registros estadísticos del Servicio de Quemados del Hospital. Como fundamentales resultados y conclusiones se obtienen las siguientes: la mayor frecuencia de casos se encontró en las edades comprendidas entre 15 y 24 años; el sexo femenino resultó ser el más afectado en todos los grupos de edades. La cocina fue el lugar donde se efectuó el acto con mayor frecuencia. Se encontró un alto grado de asociación entre la supervivencia y la presencia de antecedentes de intentos suicidas. Presentaron la mayor incidencia por municipios los de Puerto Padre y Jobabo; el alcohol y el kerosene fueron los agentes causales más utilizados. En la casuística predominaron los grandes quemados.A transversal epidemiological study of the cases of self-aggression by burns, who received medical attention at the Emergency Department of the Burns Service of the "Dr. Ernesto Guevara de la Serna" Hospital from February, 1993 to February, 1994, was carried out aimed at determining the behaviour of some variables of interest in these cases. Fifty seven cases were studied and the information was collected from the general graph of the burn patient (Model 71-05, and from the statistical records of the Burns Service of the hospital. The main results and conclusions reached are the following: the highest frequency of cases was found in ages between 15 and 24; women were the most affected in all age groups; and kitchen was the place where the

  18. Nefrectomía simple por puerto único (LESS) asistida por robot (da Vinci)

    OpenAIRE

    CASTILLO C,OCTAVIO A; VIDAL M,IVAR; SEPÚLVEDA T,FRANCISCO

    2011-01-01

    Introducción: La cirugía mínimamente invasiva en urología avanza rápidamente y la cirugía laparo-endoscópica a través de puerto único (LESS) no es la excepción. Esta técnica por vía laparoscópica presenta mucha dificultad y requiere de un cirujano laparoscópico experimentado debido a la falta de triangulación y el cruce de los instrumentos. Los beneficios del sistema quirúrgico da Vinci® han sido introducidos recientemente en LESS. Presentamos dos casos de nefrectomía LESS asistida por robot....

  19. Detection of Mycobacterium tuberculosis in extrapulmonary biopsy samples using PCR targeting IS6110, rpoB, and nested-rpoB PCR Cloning.

    Science.gov (United States)

    Meghdadi, Hossein; Khosravi, Azar D; Ghadiri, Ata A; Sina, Amir H; Alami, Ameneh

    2015-01-01

    Present study was aimed to examine the diagnostic utility of polymerase chain reaction (PCR) and nested PCR techniques for the detection of Mycobacterium tuberculosis (MTB) DNA in samples from patients with extra pulmonary tuberculosis (EPTB). In total 80 formalin-fixed, paraffin-embedded (FFPE) samples comprising 70 samples with definite diagnosis of EPTB and 10 samples from known non- EPTB on the basis of histopathology examination, were included in the study. PCR amplification targeting IS6110, rpoB gene and nested PCR targeting the rpoB gene were performed on the extracted DNAs from 80 FFPE samples. The strong positive samples were directly sequenced. For negative samples and those with weak band in nested-rpoB PCR, TA cloning was performed by cloning the products into the plasmid vector with subsequent sequencing. The 95% confidence intervals (CI) for the estimates of sensitivity and specificity were calculated for each method. Fourteen (20%), 34 (48.6%), and 60 (85.7%) of the 70 positive samples confirmed by histopathology, were positive by rpoB-PCR, IS6110-PCR, and nested-rpoB PCR, respectively. By performing TA cloning on samples that yielded weak (n = 8) or negative results (n = 10) in the PCR methods, we were able to improve their quality for later sequencing. All samples with weak band and 7 out of 10 negative samples, showed strong positive results after cloning. So nested-rpoB PCR cloning revealed positivity in 67 out of 70 confirmed samples (95.7%). The sensitivity of these combination methods was calculated as 95.7% in comparison with histopathology examination. The CI for sensitivity of the PCR methods were calculated as 11.39-31.27% for rpoB-PCR, 36.44-60.83% for IS6110- PCR, 75.29-92.93% for nested-rpoB PCR, and 87.98-99.11% for nested-rpoB PCR cloning. The 10 true EPTB negative samples by histopathology, were negative by all tested methods including cloning and were used to calculate the specificity of the applied methods. The CI for 100

  20. Consumo Voluntario de Forraje por Rumiantes en Pastoreo Consumo Voluntario de Forraje por Rumiantes en Pastoreo

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    José Mejía Haro

    2012-02-01

    Full Text Available The variation in voluntary forage intake is undoubtedly the major dietary factor determining level and efficiency of ruminant production. This variation is bigger and least predictable for grazing ruminants. Range ruminant productivity and efficiency is relatively low due, partly, to intake limitations; productivity could probably be increased most by increasing intake. Distension of the reticulo-rumen wall is the primary intake regulation mechanism of low-quality roughages in range ruminants, digestibility and rate of ingest passage also affect voluntary intake. Body size and metabolic bodysize as well affect intake of grazing animals. Kind and amount of supplementation, forage availability, and grazing intensity have been related to voluntary forage intake. La variación en el consumo voluntario de forraje es indudablemente el principal factor dietario que determina el nivel y eficiencia de producción en un rumiante. Esta variación es mayor y muy difícil de predecir bajo condiciones de pastoreo. La productividad y eficiencia de rumiantes en pastoreo es relativamente baja debido, en parte, a las limitaciones en el consumo; la productividad probablemente se podrá incrementar si se incrementa el consumo. La distensión de la pared del rumenretículo es el principal mecanismo de regulación del consumo de forrajes de baja calidad en rumiantes en pastoreo, aunque la digestibilidad y la tasa de pasaje también afectan el consumo voluntario. Igualmente, el consumo se ve afectado por el tamaño corporal y peso metabólico del animal, por la cantidad y tipo de suplemento ofrecido, por la disponibilidad de forraje y por la intensidad del pastoreo.

  1. Alteraciones bioquímicas como marcadores predictores de gravedad en pacientes con fiebre por dengue

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    Luis Ángel Villar-Centeno

    2013-08-01

    Full Text Available Introducción. El dengue es la infección transmitida por mosquitos más importante en el mundo. Existe información de que las alteraciones bioquímicas pueden utilizarse como herramientas predictoras de gravedad del dengue. Objetivo. Evaluar las alteraciones bioquímicas como posibles marcadores predictores de gravedad del dengue. Materiales y métodos. Se llevó a cabo un estudio de casos y controles anidado en una cohorte. Se seleccionaron al azar 125 casos con dengue grave y 120 controles con dengue no grave para evaluar los niveles séricos de lactato-deshidrogenasa (LDH, creatina cinasa (CK, proteína C reactiva(PCR y albúmina, en sueros obtenidos en las primeras horas de la enfermedad. Para evaluar el valor diagnóstico de cada biomarcador, se establecieron puntos de corte con una sensibilidad del 90 % enla detección de casos graves. Resultados. Se observó una asociación entre los niveles de PCR por debajo de 9,8 mg/l (OR=0,04;IC95%=0,02-0,08; p=0,000, de LDH inferiores a 400 U/L (OR=0,49; IC95%=0,24-1,02; p=0,053 y de albúmina menor de 4 mg/dl (OR=3,46; IC95%=1,96-6,12; p=0,000, con la gravedad del dengue. En contraste, los niveles de la CK no mostraron asociación con la gravedad de la enfermedad. Conclusiones. Los hallazgos de nuestro estudio sugieren una asociación de los niveles de PCR, LDH y albúmina con la gravedad del dengue. Estas pruebas bioquímicas podrían ser utilizadas como herramientas predictoras del curso clínico de la infección.   doi: http://dx.doi.org/10.7705/biomedica.v33i0.732

  2. MULTIPLEX SYBR® GREEN-REAL TIME PCR (qPCR ASSAY FOR THE DETECTION AND DIFFERENTIATION OF Bartonella henselae AND Bartonella clarridgeiae IN CATS

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    Rodrigo Staggemeier

    2014-04-01

    Full Text Available A novel SYBR® green-real time polymerase chain reaction (qPCR was developed to detect two Bartonella species, B. henselae and B. clarridgeiae, directly from blood samples. The test was used in blood samples obtained from cats living in animal shelters in Southern Brazil. Results were compared with those obtained by conventional PCR targeting Bartonella spp. Among the 47 samples analyzed, eight were positive using the conventional PCR and 12 were positive using qPCR. Importantly, the new qPCR detected the presence of both B. henselae and B. clarridgeiae in two samples. The results show that the qPCR described here may be a reliable tool for the screening and differentiation of two important Bartonella species.

  3. Estandarización de una prueba de PCR para la detección de Brucella sp.

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    Carlos Padilla R

    2003-04-01

    Full Text Available Objetivo: Estandarizar una prueba de PCR para la detección de Brucella spp. Materiales y métodos: Se usó oligonucleótidos reportados que amplifican la secuencia de 16S rRNA de Brucella spp. Fueron evaluados dos métodos de extracción de ADN: fenol-cloroformo-alcohol isoamílico y un kit comercial basado en columnas con afinidad. Para determinar la sensibilidad de la prueba se usó 8 cepas peruanas de Brucella y para determinar la especificidad de la prueba se usó otras cepas bacterianas peruanas de E. coli, Shigella, Proteus mirabilis, Salmonella aratyphi, Salmonella typhi, Citrobacter freundii y Vibrio cholerae. Resultados: Los 2 métodos de extracción de ADN evaluados fueron efectivos. La sensibilidad analítica de la prueba es alta, lográndose detectar 80 femtogramos de ADN de Brucella spp. purificado. Todas las cepas peruanas de Brucella spp. fueron detectadas por la prueba. Además, la prueba es negativa para cepas peruanas de otras especies bacterianas. Conclusión: Se ha estandarizado las condiciones de una prueba de PCR para la detección de cepas peruanas de Brucella spp., la cual es muy sensible y específica en el laboratorio.

  4. Data Analysis of Sequences and qPCR for Microbial Communities during Algal Blooms

    Science.gov (United States)

    A training opportunity is open to a highly microbial-research-motivated student to conduct sequence analysis, explore novel genes and metabolic pathways, validate resultant findings using qPCR/RT-qPCR and summarize the findings

  5. Real-Time PCR in Clinical Microbiology: Applications for Routine Laboratory Testing

    Science.gov (United States)

    Espy, M. J.; Uhl, J. R.; Sloan, L. M.; Buckwalter, S. P.; Jones, M. F.; Vetter, E. A.; Yao, J. D. C.; Wengenack, N. L.; Rosenblatt, J. E.; Cockerill, F. R.; Smith, T. F.

    2006-01-01

    Real-time PCR has revolutionized the way clinical microbiology laboratories diagnose many human microbial infections. This testing method combines PCR chemistry with fluorescent probe detection of amplified product in the same reaction vessel. In general, both PCR and amplified product detection are completed in an hour or less, which is considerably faster than conventional PCR detection methods. Real-time PCR assays provide sensitivity and specificity equivalent to that of conventional PCR combined with Southern blot analysis, and since amplification and detection steps are performed in the same closed vessel, the risk of releasing amplified nucleic acids into the environment is negligible. The combination of excellent sensitivity and specificity, low contamination risk, and speed has made real-time PCR technology an appealing alternative to culture- or immunoassay-based testing methods for diagnosing many infectious diseases. This review focuses on the application of real-time PCR in the clinical microbiology laboratory. PMID:16418529

  6. Apparatus, System and Method for Fast Detection of Genetic Information by PCR in an Interchangeable Chip

    KAUST Repository

    Wen, Weijia; Wu, Jinbo; Kodzius, Rimantas

    2011-01-01

    A polymerase chain reaction (PCR) device for fast amplification and detection of DNA includes an interchangeable PCR chamber, a temperature control component, and an optical detection system. The DNA amplification is performed on an interchangeable

  7. Intoxicación ocupacional por mercurio

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    Augusto V Ramírez

    2008-03-01

    Full Text Available El mercurio, metal pesado ampliamente utilizado por el hombre, es muy tóxico; produce daño al sistema nervioso central, perturbaciones del comportamiento y lesiones renales. Se acumula en todos los seres vivos y no es esencial para ningún proceso biológico. La toxicidad del mercurio está directamente relacionada con su estado químico. El metilmercurio es la forma más dañina, con efectos neurotóxicos en adultos y en fetos de madres expuestas. El mercurio metálico no es menos tóxico. Las sales de mercurio inorgánico afectan directamente al riñón. Clínicamente, en la exposición ocupacional a mercurio se encuentra la triada clásica: temblor, alteración de la personalidad y estomatitis. En los últimos años se ha demostrado también alteración en la visión cromática. La exposición aguda se evalúa midiendo el mercurio en la sangre, mientras que la exposición crónica y ocupacional se determina mejor dosándolo en orina homogenizada de 24 horas. Los quelantes del metal -BAL, sus derivados o la D-penicilamina- son usados para tratar la intoxicación aguda o crónica.

  8. Histeria ainda hoje, por quê?

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    Dayse Santos Costa

    2016-04-01

    Full Text Available Resumo Estudo teórico que teve o propósito de discutir a histeria na contemporaneidade, considerando as mudanças culturais que podem ter acorrido desde a fundação da psicanálise até hoje. A discussão teve como cerne os apontamentos de Charles Melman, posto que seja um psicanalista que vem pensando a posição do sujeito nas condições da cultura ocidental atual. Foram destacados aspectos que nos conduziram a elaborar um pensamento em torno de como a neurose histérica aparece no contexto contemporâneo. Partimos do princípio de que sofremos uma mutação cultural, na qual passamos de uma cultura propensa à neurose para uma propensa à perversão. Tal concepção determina que lidamos com sujeitos que funcionam sob a ordem de uma nova economia psíquica e que em decorrência assistimos a expressão de uma histeria coletiva que, por sua vez, seria espaço de reivindicação dos sujeitos, a fim de requisitar um paradeiro e reinventar um pai que já esteja destituído.

  9. Identidad e interculturalidad: un horizonte por visitar

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    Marian Pérez Bernal

    2016-01-01

    Full Text Available En el presente trabajo mantenemos que el concepto de identidad defendido por Amin Maalouf puede ser una interesante vía para conseguir una aproximación entre las distintas culturas capaz de conducirnos a un diálogo fructífero que nos permitan gestionar la multiculturalidad actual. Defendemos que las culturas sean vistas como escenas de debate y contestación en constante proceso de construcción y reconstrucción y en las que siempre primen unos valores que nos conciernen a todos y que han de ser irrenunciables.In this paper, it is stated that the concept of identity defended by Amin Maalouf could be an interesting way to achieve a rapprochement amongst different cultures. This could lead to a fruitful dialog allowing to manage today’s multiculturalism. It is also defended that cultures should be considered as instruments of debate and reply on a continuous process of construction and reconstruction. However, cultures should not prevail over a series of values that concern to all of us and that should be inalienable.

  10. Libertad, igualdad, sostenibilidad. Por un ecofeminismo ilustrado

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    Puleo, Alicia H.

    2008-06-01

    Full Text Available This article presents the proposal of an enlightened Ecofeminism and defines it as an ethic-political thinking about human relations to Nature. It aims at ecojustice and sustainability, which are characterized through the criticism of prejudice, through the principles of equality and autonomy, and through the nominalist conceptualization of gender, intercultural dialogue and the sensible acceptance of science and technique. The enlightened Ecofeminism universalizes virtues such as caring for the human and non-human Nature, and defends a compassionate moral towards the radical finitude of the world.

    En este artículo se presenta la propuesta de un ecofeminismo ilustrado entendido como reflexión ético-política sobre las relaciones de los humanos con la Naturaleza. Orientado a la ecojusticia y la sostenibilidad, el ecofeminismo ilustrado se caracterizaría por la crítica al prejuicio, la defensa de los principios de igualdad y autonomía, la conceptualización nominalista del género, el diálogo intercultural, la aceptación prudente de la ciencia y la técnica, la universalización de las virtudes del cuidado aplicadas a los humanos y al resto de la Naturaleza, y una moral de la compasión frente a la radical finitud del mundo.

  11. Estudio electrofisiologico en la neuropatia por Vincristina

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    Olga P. Sanz

    1975-12-01

    Full Text Available Diez pacientes afectados por diversas patologías que requerían tratamiento crónico con Vincristina, fueron sometidos a estudios electrofisiológicos en los que se valoró: el número de unidades motoras (UM funcionantes en los músculos de la eminencia tenar, los valores de los incrementos medios de UM, velocidad de conducción motora y su latencia residual en el nervio mediano, la velocidad de conducción sensitiva del mismo nervio y el estado de la transmisión neuromuscular. Los valores obtenidos fueron comparados con grupos controles. Los resultados mostraron disminución del número de UM; las UM remanentes presentaron amplitud reducida junto a otras cuyo tamaño no superaba el del grupo control, hecho que sugiere la incapacidad de lograr una reinervación adecuada. Las velocidades de conducción motora y sensitiva mostraron valores diminuídos, con mayor compromiso en los segmentos distales. Junto a estos datos se halló respuesta miasteniforme al estímulo repetitivo. Todos estos resultados permiten postular la existencia de un compromiso de la unidad motora, abarcando todos sus segmentos, en pacientes intoxicados con Vincristina.

  12. Comparative Evaluation Of Conventional Rt-pcr And Real-time Rt-pcr (rrt-pcr) For Detection Of Avian Metapneumovirus Subtype A [comparação Entre As Técnicas De Rt-pcr Convencional E Rt-pcr Em Tempo Real Para A Detecção Do Metapneumovírus Aviários Subtipo A

    OpenAIRE

    Ferreira H.L.; Spilki F.R.; dos Santos M.M.A.B.; de Almeida R.S.; Arns C.W.

    2009-01-01

    Avian metapneumovirus (AMPV) belongs to Metapneumovirus genus of Paramyxoviridae family. Virus isolation, serology, and detection of genomic RNA are used as diagnostic methods for AMPV. The aim of the present study was to compare the detection of six subgroup A AMPV isolates (AMPV/A) viral RNA by using different conventional and real time RT-PCR methods. Two new RT-PCR tests and two real time RT-PCR tests, both detecting fusion (F) gene and nucleocapsid (N) gene were compared with an establis...

  13. Reação em Cadeia da Polimerase (PCR baseada no gene cpx para detecção de Actinobacillus pleuropneumoniae em suínos natural e experimentalmente infectados Polymerase Chain Reaction (PCR based on the cpx gene for detection of Actinobacillus pleuropneumoniae in natural and experimentally infected pigs

    Directory of Open Access Journals (Sweden)

    Karina Koerich de Souza

    2008-10-01

    Full Text Available A pleuropneumonia suína é uma das mais importantes doenças respiratórias dos suínos, estando presente em todos os países produtores. Para o controle e o monitoramento da pleuropneumonia, é necessário o desenvolvimento de métodos rápidos e acurados de diagnóstico. Com o objetivo de validar a técnica da PCR, baseada no gene cpx de Actinobacillus pleuropneumoniae, em suínos sabidamente positivos, primeiramente foi realizada inoculação experimental com amostras de A. pleuropneumoniae sorotipo 5B e coletadas amostras por meio de suabe de tonsila, biópsia de tonsila e sangue para realização da técnica de PCR, isolamento bacteriológico e teste de ELISA, respectivamente. Posteriormente, estas técnicas foram aplicadas em suínos naturalmente infectados, em três rebanhos com diferentes situações sanitárias quanto à apresentação clínica da doença. De cada rebanho, foram analisados cinco grupos de suínos com idades diferentes, sendo coletado de cada animal biópsia de tonsila para isolamento bacteriológico e PCR e sangue para determinação do perfil sorológico. Os resultados obtidos na inoculação experimental confirmaram que, mesmo com o estabelecimento da infecção comprovada pelo isolamento bacteriológico, após o período de 45 dias, não foi possível detectar o agente pela técnica de PCR. Em animais naturalmente infectados, a técnica de PCR apresentou maior sensibilidade quando comparado com o isolamento. A associação entre PCR e ELISA demonstrou ser uma boa alternativa para definir a situação sanitária do rebanho quanto à infecção por A. pleuropneumoniae.Swine pleuropneumonia is one of the most important pig respiratory diseases and has been found in all producer countries. For control and monitoring of pleuropneumonia, it is necessary the development of fast and specific methods of diagnosis. To validate PCR based on the cpx gene of Actinobacillus pleuropneumoniae in positive pigs, an experimental

  14. The PCR-GLOBWB global hydrological reanalysis product

    Science.gov (United States)

    Wanders, Niko; Bierkens, Marc; Sutanudjaja, Edwin; van Beek, Rens

    2014-05-01

    Accurate and long time series of hydrological data are important for understanding land surface water and energy budgets in many parts of the world, as well as for improving real-time hydrological monitoring and climate change anticipation. The ultimate goal of the present work is to produce a multi-decadal "land surface hydrological reanalysis" dataset with retrospective and updated hydrological states and fluxes that are constrained to available in-situ river discharge measurements. Here we use PCR-GLOBWB (van Beek et al., 2011), which is a large-scale hydrological model intended for global to regional studies. PCR-GLOBWB provides a grid-based representation of terrestrial hydrology with a typical spatial resolution of approximately 50×50 km (currently 0.5° globally) on a daily basis. For each grid cell, PCR-GLOBWB simulates moisture storage in two vertically stacked soil layers as well as the water exchange between the soil and the atmosphere and the underlying groundwater reservoir. Exchange to the atmosphere comprises precipitation, evaporation and transpiration, as well as snow accumulation and melt, which are all simulated by considering vegetation phenology and sub-grid variations of elevation, land cover and soil saturation distribution. The model includes improved schemes for runoff-infiltration partitioning, interflow, groundwater recharge and baseflow, as well as river routing of discharge. It also dynamically simulates water storage in reservoirs, water demand and the withdrawal, allocation and consumptive use of surface water and groundwater resources. By embedding the PCR-GLOBWB model in an Ensemble Kalman Filter framework, we calibrate the model parameters based on the discharge observations from the Global Runoff Data Centre. The parameters calibrated are related to snow accumulation and melt, runoff-infiltration partitioning, groundwater recharge, channel discharge and baseflow processes, as well as pre-factors to correct forcing precipitation

  15. BROTES POR NOROVIRUS EN RESIDENCIAS Y CENTROS SANITARIOS DE CATALUÑA

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    Pere Godoy

    2009-01-01

    Full Text Available Fundamentos. La baja dosis infectiva y las múltiples vías de transmisión facilitan la presentación de brotes de norovirus. El objetivo fue comparar la incidencia de brotes por norovirus en hospitales y residencias en Cataluña. Métodos. Se realizó un estudio descriptivo de la serie de brotes de norovirus en el período del 15/10/2004 al 30/10/2005. Se rellenó una encuesta epidemiológica para cada brote. Las variables fueron: número de expuestos, enfermos, mecanismo de transmisión, ámbito (centros sanitarios o residencias, región sanitaria, mes del año, y duración. Mediante técnicas de PCR se investigó la presencia de norovirus en muestras clínicas. Se calculó la incidencia en cada centro y la incidencia anual de brotes por centros. Las diferencias se determinaron con la prueba de c2 y la t de Student con un grado de significación (p inferior a 0,05. Resultados. Se detectaron 17 brotes, 6 en centros sanitarios y 11 en residencias. La tasa de ataque global fue del 33,4% (652/1951 y fue ligeramente superior en las residencias (35,2% que en los centros sanitarios (31,4%. El 94,1% (16/17 de los brotes se produjeron por transmisión persona a persona y sólo el 5,9% (1/17 por alimentos. La media de días entre el primer y último caso del brote fue de 11,4 (DE = 6,9. La duración media de los síntomas fue de 2,39 días (SD=1,6 y también fue superior en los pacientes hospitalizados 2,63 (SD=1,7 en comparación a los pacientes de residencias 1,97 (SD=1,7 (p < 0,0001. Conclusiones. Norovirus es responsable de un número importante de brotes por transmisión persona a persona. Se debe protocolizar su control para reducir su número y su duración.

  16. Diagnosis of lymphoma in paraffin wax sections by nested PCR and immunohistochemistry.

    OpenAIRE

    Kitamura, Y; Nanba, E; Inui, S; Tanigawa, T; Ichihara, K

    1996-01-01

    AIMS: To investigate whether nested polymerase chain reaction (PCR) and immunohistochemistry can be used to diagnose malignant lymphoma. METHODS: Paraffin wax embedded tissue sections from 31 patients with malignant lymphoma were analysed by nested PCR and immunohistochemistry using standard protocols. RESULTS: Nested PCR amplification of 1 pg DNA confirmed monoclonality in B cell lymphoma; PCR amplification of 10 pg DNA confirmed monoclonality in T cell lymphoma. Twenty seven (87%) samples w...

  17. Detection and Analysis of Circular RNAs by RT-PCR.

    Science.gov (United States)

    Panda, Amaresh C; Gorospe, Myriam

    2018-03-20

    Gene expression in eukaryotic cells is tightly regulated at the transcriptional and posttranscriptional levels. Posttranscriptional processes, including pre-mRNA splicing, mRNA export, mRNA turnover, and mRNA translation, are controlled by RNA-binding proteins (RBPs) and noncoding (nc)RNAs. The vast family of ncRNAs comprises diverse regulatory RNAs, such as microRNAs and long noncoding (lnc)RNAs, but also the poorly explored class of circular (circ)RNAs. Although first discovered more than three decades ago by electron microscopy, only the advent of high-throughput RNA-sequencing (RNA-seq) and the development of innovative bioinformatic pipelines have begun to allow the systematic identification of circRNAs (Szabo and Salzman, 2016; Panda et al ., 2017b; Panda et al ., 2017c). However, the validation of true circRNAs identified by RNA sequencing requires other molecular biology techniques including reverse transcription (RT) followed by conventional or quantitative (q) polymerase chain reaction (PCR), and Northern blot analysis (Jeck and Sharpless, 2014). RT-qPCR analysis of circular RNAs using divergent primers has been widely used for the detection, validation, and sometimes quantification of circRNAs (Abdelmohsen et al ., 2015 and 2017; Panda et al ., 2017b). As detailed here, divergent primers designed to span the circRNA backsplice junction sequence can specifically amplify the circRNAs and not the counterpart linear RNA. In sum, RT-PCR analysis using divergent primers allows direct detection and quantification of circRNAs.

  18. Directed PCR-free engineering of highly repetitive DNA sequences

    Directory of Open Access Journals (Sweden)

    Preissler Steffen

    2011-09-01

    Full Text Available Abstract Background Highly repetitive nucleotide sequences are commonly found in nature e.g. in telomeres, microsatellite DNA, polyadenine (poly(A tails of eukaryotic messenger RNA as well as in several inherited human disorders linked to trinucleotide repeat expansions in the genome. Therefore, studying repetitive sequences is of biological, biotechnological and medical relevance. However, cloning of such repetitive DNA sequences is challenging because specific PCR-based amplification is hampered by the lack of unique primer binding sites resulting in unspecific products. Results For the PCR-free generation of repetitive DNA sequences we used antiparallel oligonucleotides flanked by restriction sites of Type IIS endonucleases. The arrangement of recognition sites allowed for stepwise and seamless elongation of repetitive sequences. This facilitated the assembly of repetitive DNA segments and open reading frames encoding polypeptides with periodic amino acid sequences of any desired length. By this strategy we cloned a series of polyglutamine encoding sequences as well as highly repetitive polyadenine tracts. Such repetitive sequences can be used for diverse biotechnological applications. As an example, the polyglutamine sequences were expressed as His6-SUMO fusion proteins in Escherichia coli cells to study their aggregation behavior in vitro. The His6-SUMO moiety enabled affinity purification of the polyglutamine proteins, increased their solubility, and allowed controlled induction of the aggregation process. We successfully purified the fusions proteins and provide an example for their applicability in filter retardation assays. Conclusion Our seamless cloning strategy is PCR-free and allows the directed and efficient generation of highly repetitive DNA sequences of defined lengths by simple standard cloning procedures.

  19. [Differentiation of geographic biovariants of smallpox virus by PCR].

    Science.gov (United States)

    Babkin, I V; Babkina, I N

    2010-01-01

    Comparative analysis of amino acid and nucleotides sequences of ORFs located in extended segments of the terminal variable regions in variola virus genome detected a promising locus for viral genotyping according to the geographic origin. This is ORF O1L of VARV. The primers were calculated for synthesis of this ORF fragment by PCR, which makes it possible to distinguish South America-Western Africa genotype from other VARV strains. Subsequent RFLP analysis reliably differentiated Asian strains from African strains (except Western Africa isolates). This method has been tested using 16 VARV strains from various geographic regions. The developed approach is simple, fast and reliable.

  20. Methods for microbial DNA extraction from soil for PCR amplification

    Directory of Open Access Journals (Sweden)

    Yeates C

    1998-01-01

    Full Text Available Amplification of DNA from soil is often inhibited by co-purified contaminants. A rapid, inexpensive, large-scale DNA extraction method involving minimal purification has been developed that is applicable to various soil types (1. DNA is also suitable for PCR amplification using various DNA targets. DNA was extracted from 100g of soil using direct lysis with glass beads and SDS followed by potassium acetate precipitation, polyethylene glycol precipitation, phenol extraction and isopropanol precipitation. This method was compared to other DNA extraction methods with regard to DNA purity and size.