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.

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.

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

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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...

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.

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.

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

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

Temporal expression pattern of myostatin transcripts during chicken embryogenesis Padrão de expressão temporal de transcritos de miostatina durante a embriogênese da galinha

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J.E. Gabriel; L.E. Alvares; C.C.P. Paz; I.U. Packer; L.L. Coutinho

2006-01-01

No presente estudo, estimou-se a abundância dos transcritos da miostatina foi estimada durante a embriogênese de galinha por análises de RT-PCR competitiva. Níveis basais de mRNA desse gene foram detectados até o estádio HH15, enquanto acúmulos significativos nesses níveis foram observados apenas no estádio HH24, seguido por redução na abundância desses transcritos a partir do estádio HH26. Tais descobertas preliminares proporcionam informações relevantes sobre a ativação do fator de crescime...

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.

Pericardite em suínos ao abate no Rio Grande Sul: avaliação de agentes bacterianos e lesões associadas

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Carolini F. Coelho

2014-07-01

Full Text Available O objetivo do presente estudo foi identificar a frequência de lesões macroscópicas e microscópicas e dos agentes bacterianos envolvidos em pericardites em suínos no abate no Estado do Rio Grande do Sul. As amostras foram coletadas em frigoríficos de suínos com Serviço de Inspeção Federal (SIF entre fevereiro a outubro de 2010 e a condenação por pericardite dos animais acompanhados foi de 3,9% (299/7.571. No total foram investigados 91 casos de pericardites, 89% deles foram classificados como crônicos por histopatologia e pleurite crônica foi observada em 47% dos pulmões correspondentes, todavia não houve associação significativa entre as duas lesões. Os agentes bacterianos isolados a partir dos corações foram Streptococcus spp., Pasteurella multocida, Haemophilus parasuis e Streptococcus suis. DNA bacterianos mais detectados pela PCR foram de Mycoplasma hyopneumoniae e Actinobacillus pleuropneumoniae. Houve associação significativa entre isolamento de P. multocida e Streptococcus sp. nos corações e pulmões correspondentes. Esses resultados sugerem que a infecção no pulmão possa ter servido de porta de entrada para a colonização do pericárdio adjacente. Apesar de M. hyopneumoniae ter sido o agente detectado com maior frequência pela PCR em corações e pulmões correspondentes, não houve associação significativa da detecção dos agentes nos órgãos. Isto sugere que as infecções foram eventos independentes. Os demais agentes investigados não apresentaram associação significativa entre isolamento ou detecção de DNA em coração e pulmão correspondente. Outro achado importante foi a presença de coinfecções bacterianas em 2% dos corações e por PCR foi detectado DNA bacteriano de dois ou mais agentes em 16,5% dos corações. Esses resultados sugerem que as coinfecções em pericardites precisam ser melhor estudadas.

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.

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

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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...

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

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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...

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.

Temporal expression pattern of myostatin transcripts during chicken embryogenesis Padrão de expressão temporal de transcritos de miostatina durante a embriogênese da galinha

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J.E. Gabriel

2006-10-01

Full Text Available No presente estudo, estimou-se a abundância dos transcritos da miostatina foi estimada durante a embriogênese de galinha por análises de RT-PCR competitiva. Níveis basais de mRNA desse gene foram detectados até o estádio HH15, enquanto acúmulos significativos nesses níveis foram observados apenas no estádio HH24, seguido por redução na abundância desses transcritos a partir do estádio HH26. Tais descobertas preliminares proporcionam informações relevantes sobre a ativação do fator de crescimento miostatina durante o desenvolvimento in ovo de aves.

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

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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.

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.

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

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

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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.

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

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...

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.

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

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

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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

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

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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

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%.

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

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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.

Celulitis por citomegalovirus

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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.

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.

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.

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

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

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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

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.

Infecciones por bacterias poco comunes y oncogénesis bacteriana

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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.

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.

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

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

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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.

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

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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.

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

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

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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.

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.

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.

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

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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.

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

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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.

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

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

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

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

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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

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.

Abortos por Neosporacaninum em bovinos do sul de Minas Gerais

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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.

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

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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

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

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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.

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

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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.

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

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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

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.

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

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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

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

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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.

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

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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

Rabdomioma cardiaco múltiple asociado a muerte intrauterina

OpenAIRE

Morales-Quispe,Jorge A.; Espínola-Zavaleta,Nilda; Caballero-Caballero,Rocío; Brunner-Cruz,Guadalupe; Uribe Alcántara,Sergio

2011-01-01

El rabdomioma es el tumor más frecuentemente detectado en los niños desde la vida fetal, aunque su incidencia es muy baja. Este tumor es histológicamente benigno, pero puede provocar repercusión hemodinámica y manifestarse con datos de bajo gasto cardiaco, arritmias y excepcionalmente con muerte intrauterina, como en el presente caso, que fue detectado por medio de ultrasonido obstétrico, ecocardiograma fetal y se corroboró con el estudio histopatológico.

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.

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

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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.

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.

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.

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.

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

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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

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.

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.

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

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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.

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

Epidemiología molecular de infección nosocomial por Klebsiella pneumoniae productora de beta-lactamasas de espectro extendido.

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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.

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

Energy Technology Data Exchange (ETDEWEB)

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)

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

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…

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.

CLONACIÓN Y FILOGENIA MOLECULAR DE UN SEGMENTO DEL GEN CODANTE DE LA ACTINA DE MYRCIARIA DUBIA “CAMU-CAMU”: UN CANDIDATO PARA GEN DE REFERENCIA

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Juan Carlos Castro Gómez

2012-12-01

Full Text Available Myrciaria dubia “camu-camu” es un frutal amazónico caracterizado por su amplia variación de vitamina C. Pero los estudios genético moleculares que puedan explicar esta variación son limitados. Por ello nuestro objetivo fue realizar la clonación y filogenia molecular de un segmento del gen codante de la actina de M. dubia. Las muestras fueron obtenidas de la colección de germoplasma del INIA. Luego, el ARN fue purificado y mediante RT-PCR con cebadores degenerados se amplificó un segmento del gen. En base a la secuencia obtenida se diseñaron cebadores específicos para PCR en tiempo real. Los resultados muestran que se ha aislado, clonado y secuenciado un segmento del gen codante de actina de M. dubia y detectado su expresión en hojas, pulpa y cáscara de M. dubia. Así, con el soporte de herramientas bioinformáticas y uso de técnicas de biología molecular hemos aislado, clonado y secuenciado un segmento del gen codante de la actina de M. dubia. Asimismo, los análisis realizados muestran que el gen se expresa y presenta niveles similares de expresión en hojas, pulpa y cáscara de M. dubia. Sin embargo, es necesario realizar más experimentos a fin de verificar su estabilidad de expresión.

Adquisición de los sujetos pronominales en español por aprendientes anglófonos

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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.

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

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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

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

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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.

Frequency of Mycoplasma hominis and Ureaplasma urealyticum infections in women with systemic lupus erythematosus Freqüência da infecção pelo Mycoplasma hominis e Ureaplasma urealyticum em mulheres portadoras de lupus eritematoso sistêmico

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Alcyone A. Machado

2001-06-01

Full Text Available Ureaplasma urealyticum (UU and Mycoplasma hominis (MH have been detected in the urine of women with systemic lupus erythematosus (SLE. We evaluated the presence of these mycoplasma in the endocervix of women presenting SLE. A total of 40 SLE patients (mean age 40.2 years, and 51 healthy women (mean age 30.9 years, were studied. Endocervical swabs were cultured in specific liquid media for MH or UU, detected by a quantitative color assay, and considered positive at >10³ dilutions. Statistical analysis was performed using the two-tailed Fisher test. UU was detected in 52.5 % of patients and in 11.8% of controls (p= 0.000059. MH was detected in 20% of patients and 2% controls (p=0.003905. Both mycoplasmas were detected in 7.3% patients and 0% controls (pUreaplasma urealyticum (UU e Mycoplasma hominis (MH têm sido detectados em urina de mulheres com lupus eritematoso sistêmico (LES. Avaliamos a presença destes mycoplasmas no endocervix de mulheres apresentando LES. Um total de 40 pacientes com LES (idade média de 40,2 anos, e 51 mulheres sadias (idade média de 30.9 anos, foram estudadas. Swabs do endocervix foram cultivados em meio líquido específico para MH e UU, detectados por teste colorimétrico quantitativo, considerando positivo diluições > 10³ . Análise estatística foi feita usando teste de Fisher. UU foi detectado em 52,5% das pacientes e em 11,8% dos controles (p= 0.000059. MH foi detectado em 20% das pacientes e 2% dos controles (p=0.003905. Ambos mycoplasmas foram detectados em 7,3 % das pacientes e 0% dos controles (p<0.000001. Os resultados aqui reportados corroboram com a associação de infecção por mycoplasma e LES. Estes agentes podem estimular a produção de clones autoreativos.

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.

Validez de la reacción en cadena de la polimerasa para el diagnóstico de infecciones respiratorias producidas por Mycoplasma pneumoniae:meta-análisis

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Olga Lucía Sarmiento

2013-09-01

Full Text Available Introducción. Mycoplasma pneumoniae es considerado un frecuente agente etiológico de infecciones respiratorias y de una amplia gama de manifestaciones extra-pulmonares. Pruebas diagnósticas con baja exactitud y validez llevaron a considerar la reacción en cadena de la polimerasa (PCR para el diagnóstico de M. pneumoniae. Objetivo: Evaluar la validez y la exactitud de la PCR para la detección de M. pneumoniae. Materiales y Métodos: Meta-análisis de estudios que evaluaron el diagnóstico de M. pneumoniae en tracto respiratorio por PCR, publicados en MEDLINE desde noviembre de 1966 hasta julio de 2009. El análisis estadístico incluyó: i cálculo de sensibilidad y especifi cidad de la PCR; ii prueba de homogeneidad de estimadores entre estudios; iii elaboración de summary receiver operating characteristic curves (SROC por un modelo de regresión lineal; iv método de Egger para evaluar sesgo de publicación. Resultados: De 44 estudios incluidos los cuales incluyeron 6111 pacientes se calculó una sensibilidad de 0,72 (rango 0,21-0,99 y una especifi cidad de 0,96 (rango 0,06-0,99. Estudios en los cuales se incluyeron solo niños la PCR mostró menor exactitud. Estudios con concentraciones de cloruro de magnesio mayores de 1,5 mM mostraron mayor exactitud. Conclusiones: Según los resultados de este estudio, la PCR no se recomienda como prueba rutinaria. Sin embargo, en casos con alta sospecha clínica de infección por M. pneumoniae en servicios de salud con alta prevalencia, la PCR es de utilidad. Estudios futuros deben reportar el espectro de la enfermedad y los resultados deben ser reportados por tipo de muestra y de acuerdo a los diferentes puntos de corte de la prueba de referencia utilizada.

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.

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...

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.

Biorremediación de arsénico mediada por microorganismos genéticamente modificados

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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

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

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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.

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

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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

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

Directory of Open Access Journals (Sweden)

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

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.

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

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

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

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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.

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

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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.

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

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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.

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.

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

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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.

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

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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.

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.

Ocorrência do subtipo B do vírus da imunodeficiência felina em gatos domésticos da região sul do estado do Rio Grande do Sul, Brasil

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F.S. Silva

2014-02-01

Full Text Available No Brasil existem poucos estudos sobre a ocorrência da infecção pelo vírus da imunodeficiência felina (FIV, assim como a determinação dos subtipos circulantes, o que é indispensável para o desenvolvimento de vacinas e novos testes diagnósticos. O presente trabalho investigou a ocorrência da infecção pelo FIV entre os anos de 2010 e 2011 em gatos domésticos submetidos a atendimento clínico na cidade de Pelotas e região. Amostras de sangue total de 70 animais, incluindo suspeitos (28 ou não suspeitos (42 da infecção pelo FIV, foram submetidas à reação de PCR nested. Os resultados indicaram uma frequência de infecção de 15,7% (11/70 e a análise dos fatores associados (sexo, idade e condição clínica evidenciou uma maior ocorrência em gatos com idade superior a 10 anos e acometidos por infecções crônicas e recidivantes. Oito amostras positivas na PCR nested foram submetidas a sequenciamento genômico e somente o subtipo B foi detectado na região estudada.

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.

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.

Screening of Passiflora species for reaction to Cowpea aphid-borne mosaic virus reveals an immune wild species Seleção de espécies de Passiflora inoculadas com o vírus do mosaico do caupi revela a imunidade de uma espécie selvagem

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Scheila da Conceição Maciel

2009-06-01

Full Text Available Cowpea aphid-borne mosaic virus (CABMV is a potyvirus that causes the most serious virus disease of passion fruit crops in Brazil. It is transmitted by several species of aphids in a non-persistent, non-circulative manner. The reaction of 16 species of Passiflora to infection by mechanical inoculation with four Brazilian isolates of CABMV was evaluated under greenhouse conditions. Only P. suberosa, a wild species, was resistant to infection by all virus isolates, in two independent assays. P. suberosa grafted onto infected P. edulis f. flavicarpa did not develop symptoms; neither was the virus detected by RT-PCR in the upper leaves, suggesting that this species is immune to CABMV.O vírus do mosaico do caupi (Cowpea aphid-borne mosaic virus - CABMV é um potyvirus que causa uma das mais importantes doenças do maracujazeiro no Brasil. O vírus é transmitido por diversas espécies de afídeos de maneira não persistente, não circulativa. A reação de 16 espécies de Passiflora à infecção com quatro isolados brasileiros do CABMV, por meio de inoculação mecânica foi avaliada em condições de casa-de-vegetação. Somente a espécie selvagem P. suberosa foi resistente à infecção com todos os isolados do CABMV, em dois ensaios independentes. Plantas de P. suberosa enxertadas em plantas de P. edulis f. flavicarpa infectadas com o CABMV também não desenvolveram sintomas da doença. O vírus também não foi detectado por RT-PCR nas folhas superiores das plantas, sugerindo que essa espécie é imune ao CABMV.

Diagnóstico de virus respiratorios utilizando un sistema automatizado de PCR múltiples (FilmArray y su comparación con métodos convencionales

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Débora N Marcone

2015-03-01

Full Text Available Las infecciones respiratorias agudas producen una importante morbimortalidad y comúnmente son causadas por virus. En Argentina, los programas de vigilancia epidemiológica se basan en la detección de antígenos virales por inmunofluorescencia (IF, aunque es bien conocido que los métodos moleculares son más sensibles. El panel respiratorio (PR FilmArray (PR-FilmArray es un equipo comercial automatizado de PCR múltiples que detecta 17 virus respiratorios y 3 bacterias, en un sistema cerrado que requiere 5 min de procesamiento y una 1 h de instrumentación. Se evaluó un total de 315 muestras respiratorias de niños menores de 6 años con infecciones respiratorias agudas por IF para 8 virus respiratorios y por RT-PCR para rinovirus. Posteriormente, estas muestras se estudiaron con el PR-FilmArray. La frecuencia de positividad al considerar los 9 virus estudiados por IF y RT-PCR fue del 75 %; por PR-FilmArray fue del 92 %. El porcentaje de acuerdo positivo entre ambas metodologías fue del 70,5 % y el de acuerdo negativo fue del 99,6 % (intervalo de confianza 95 %: 65,5-75,1 y 99,2-99,8, respectivamente. El PR-FilmArray permitió obtener un mayor diagnóstico positivo (97 % y detectó otros virus, como los coronavirus NL63, 229E, OC43 y HKU1 (10 % y los bocavirus (18 %. Además, permitió identificar coinfecciones múltiples (39 % con 2, 3, 4 y hasta 5 virus. Actualmente, la IF continúa siendo el método más utilizado en los países latinoamericanos para el diagnóstico de virus respiratorios por su bajo costo, por su capacidad para procesar un alto número de muestras simultáneamente y porque los resultados de los virus más frecuentes están disponibles en 5 h. Sin embargo, la futura incorporación de métodos moleculares aumentaría notablemente la capacidad diagnóstica.

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

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

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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.

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

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

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

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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.

Rinovirus: Frecuencia en niños con infección respiratoria aguda, no internados

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Débora N. Marcone

2012-02-01

Full Text Available Los métodos moleculares para diagnosticar rinovirus humanos (RVH han aumentado la sensibilidad de detección. Esto ha permitido documentar la asociación entre los RVH y las infecciones respiratorias agudas (IRA altas y bajas. La infección por RVH durante la infancia se asoció con posterior desarrollo de asma. Se estudió la frecuencia de RVH en 186 niños menores de 6 años ambulatorios con IRA (alta o baja, durante 2 años consecutivos (1/6/2008 - 31/5/2010. Se correlacionó la presencia de RVH con los antecedentes y características clínico-epidemiológicas. La detección de RVH se realizó con una RT-PCR en tiempo real que amplifica parte de la región 5' no codificante del genoma. Los virus respiratorios clásicos se estudiaron por inmunofluorescencia. En el 61% de los niños se detectó etiología viral. Las frecuencias fueron: RVH 27%, virus sincicial respiratorio (VSR 16%, influenza A y B 9%, parainfluenza 8%, metapneumovirus 7% y adenovirus 0.5%. Se observaron coinfecciones duales en 8 casos, siendo RVH el más frecuente (en 4 de ellos. Los RVH circularon durante todo el período estudiado, con picos en invierno y primavera. No se observaron diferencias clínico-epidemiológicas significativas entre pacientes con o sin RVH, excepto un mayor porcentaje de niños afebriles con RVH. Los RVH fueron los virus más detectados en niños ambulatorios, principalmente en menores de 2 años, los segundos virus asociados a bronquiolitis, luego del VSR, y detectados tres veces más en los niños expuestos a tabaquismo pasivo (OR: 2,91; p = 0.012 que en el resto. Fueron identificados como único agente en el 28% de las bronquiolitis.

Evidência molecular da ocorrência de um fitoplasma associado ao lenho mole da macieira Molecular evidence for an association of a phytoplasma with apple rubbery wood

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Luiz Fernando Caldeira Ribeiro

2007-03-01

Full Text Available O lenho mole da macieira é uma doença relevante em diversas partes do mundo. Sintomas típicos desta doença têm sido observados em pomares instalados em estados do sul do território brasileiro desde a década de oitenta. Enxertia tem revelado a natureza infecciosa da doença e a observação de corpúsculos filamentosos no floema tem evidenciado possível associação com fitoplasma. No presente trabalho plantas com sintomas de lenho mole foram coletadas em pomar comercial, visando demonstrar a presença de fitoplasma em tecido doente, bem como identificar molecularmente este fitoplasma. Através do emprego de duplo PCR com iniciadores universais R16mF2/R1 e R16F2n/R2, fitoplasma foi consistentemente detectado em plantas sintomáticas. A identificação conduzida com duplo PCR usando-se iniciadores específicos R16(IIIF2/R demonstrou que o fitoplasma detectado pertencia ao grupo 16SrIII. Análises de RFLP conduzidas com as endonucleases AluI, KpnI, HinfI, HpaII, MseI, RsaI e SauIIIA confirmaram que o fitoplasma era um representante típico do grupo 16SrIII. A detecção e identificação molecular se constitui numa forte evidência que um fitoplasma está associado ao lenho mole da macieira no Brasil, complementando os trabalhos realizados anteriormente com transmissão por enxertia e observação por microscopia eletrônica .Apple rubbery wood is an important disease occurring worldwide. Typical symptoms have been observed since 80' decade in orchards located in the South part of Brazil. In previous studies, grafting has evidenciated that the disease had infeccious etiology and visualization of filamentous bodies inside phloem had indicated that a phytoplasma could be associated with the disease. In the present study, plants with symptoms of rubbery wood were sampled in a commercial orchard in order to demonstrate the presence of phytoplasma in infected tissue and to identify molecularly that the organism. Using nested PCR with universal

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

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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.

Controle interno da qualidade em citopatologia ginecológica: um estudo de 48.355 casos

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Arcuri Roberto Alfonso

2002-01-01

Full Text Available Todos os sistemas de avaliação de desempenho em laboratórios de citopatologia exigem um programa de controle interno da qualidade estabelecido e executado. O presente estudo tem por objetivo identificar os resultados do programa e o desempenho dos citotécnicos. Para tanto foram revistos os laudos, emitidos pelos citotécnicos e pelos citopatologistas, de 48.355 exames citopatológicos ginecológicos realizados durante 21 meses. Foram identificados 2.299 casos anômalos encaminhados ao citopatologista, e confirmados 1.132; 2.973 casos de alto risco foram detectados e 998 anômalos identificados. A revisão de 10% dos negativos representou 3.180 casos, com identificação de 77 anômalos. Os anômalos totais detectados foram: 1.176 Ascus, 60 Agus, 819 Nic I/HPV, 85 Nic II, 33 Nic III, 11 carcinomas escamosos, 17 adenocarcinomas, cinco Vain I e um Vain III. Os Ascus/Agus foram 56%, e as outras lesões representaram 44%. Os 4,56% de anômalos detectados concordam com os valores propostos por Cardin (3% a 5%, pelo sistema Bethesda (4% a 6% e por Kurman et al. (5% (4, 6, 7. O citopatologista revisou 17,48% dos casos (n = 8.452. Dos 77 (0,16% do total anômalos identificados no reescrutínio, 59 (0,12% foram Ascus, seis (0,01% Agus e 12 (0,02% Nic I. O presente estudo mostra resultados do controle interno da qualidade desenvolvido em laboratório particular e fornece dados relevantes para o planejamento de um programa de prevenção do câncer uterocervical.

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.)

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

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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.

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

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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.

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

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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.

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.

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

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

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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.

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

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.

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

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

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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.

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

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

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

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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

Brazilian avian metapneumovirus subtypes A and B: experimental infection of broilers and evaluation of vaccine efficacy Metapneumovirus aviário subtipos A e B brasileiros: infecção experimental em frangos de corte e eficácia vacinal

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Márcia B. dos Santos

2012-12-01

com os mesmos isolados. Após o desafio experimental demonstrou-se, por isolamento viral e PCR em tempo real, que o isolado do subtipo B replicou por maior período de tempo e em quantidades maiores, em comparação com o subtipo A. Para o estudo de proteção, 18 pintos de um dia de idade foram vacinados e desafiados aos 21 dias. Usando isolamento viral e PCR em tempo real, em nenhuma ave vacinada e desafiada com aMPV/A foi detectado o vírus, ao passo que uma ave vacinada e desafiada com o aMPV/B foi positiva. Os resultados mostraram que a vacina do subtipo B forneceu protecção heteróloga completa, embora a protecção homóloga não tenha sido conferida em uma ave. Apesar de o aMPV/B ter sido detectado em apenas um frango após vacinação homóloga, a replicação viral em aves vacinadas pode resultar em emergência de mutantes de escape.

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

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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%.

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...

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.

Real-time PCR in virology.

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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.

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

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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

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

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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.

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

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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

Investigação molecular de Ehrlichia spp. e Anaplasma platys em felinos domésticos: alterações clínicas, hematológicas e bioquímicas

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Elisabete S Correa

2011-10-01

Full Text Available Ehrlichia sp. e Anaplasma platys são micro-organismos Gram negativos, parasitos intracelulares obrigatórios, residindo em vacúolos citoplasmáticos de leucócitos e plaquetas, encontrados no sangue periférico ou em tecidos. Poucos relatos têm sido feitos sobre erliquiose e anaplasmose em gatos no Brasil, os quais são baseados na presença de mórulas em leucócitos e plaquetas, ou pela detecção de anticorpos. O objetivo deste trabalho foi investigar a infecção natural por Ehrlichia sp. e A.platys em gatos no Município de Campos dos Goytacazes-RJ, através da hematoscopia e pela detecção do DNA desses agentes. Foram utilizadas amostras de sangue total e de soro de 91 gatos, independente de raça, sexo e idade. Realizaram-se hemograma, bioquímica sérica e PCR, utilizando oligonucleotídes para Ehrlichia sp. e A.platys. Os dados de hematoscopia mostraram que 9,89% dos gatos apresentaram mórulas em macroplaquetas. O DNA de A.platys foi detectado em 13,18% dos 91 animais e em 44,44% das amostras positivas à hematoscopia. O DNA de Ehrlichia sp. não foi detectado em nenhuma amostra. Nenhuma alteração foi observada nos sinais clínicos nem nos resultados laboratoriais nos animais estudados. Os dados sugerem que os felinos domésticos podem atuar como potenciais reservatórios para A. platys, como forma não sintomática das enfermidades relacionadas

Caracterização genética de cepas de Yersinia pestis

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BARROS, Maria Paloma Silva de

2012-01-01

A peste é uma doença que permanece enraizada em inúmeros focos naturais por todo o mundo. Embora o Brasil passe por um período de silenciamento epidemiológico, anticorpos antipestosos são detectados nas atividades de vigilância, sugerindo que estes focos permanecem ativos. A Yersinia pestis, agente causador da peste, apresenta uma história evolutiva recente e é considerada uma espécie, geneticamente, muito homogênea. Diante da necessidade de estudos mais aprofundados sobre a...

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

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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

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

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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.

Detection of putative periodontal pathogens in subgingival specimens of dogs Detecção de patógenos periodontais em amostras subgengivais de cães

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Sheila Alexandra Belini Nishiyama

2007-03-01

Full Text Available In this study, the presence of putative periodontal organisms, Porphyromonas gingivalis, Prevotella intermedia, Tannerella forsythensis, Fusobacterium nucleatum,Dialister pneumosintes,Actinobacillus actinomycetemcomitans,Campylobacter rectus,Eikenella corrodens and Treponema denticola were examined from subgingival samples of 40 dogs of different breeds with (25 and without (15 periodontitis, by using the PCR method. The PCR products of each species showed specific amplicons. Of the 25 dogs with periodontitis, P. gingivalis was detected in 16 (64% samples, C. rectus in 9 (36%, A. actinomycetemcomitans in 6 (24%, P. intermedia in 5 (20%, T. forsythensis in 5 (20%, F. nucleatum in 4 (16% and E. corrodens in 3 (12%. T. denticola and D. pneumosintes were not detected in clinical samples from dogs with periodontitis. Moreover, P. gingivalis was detected only in one (6.66% crossbred dog without periodontitis. Our results show that these microorganisms are present in periodontal microbiota of dogs with periodontitits, and it is important to evaluate the role of these putative periodontal microorganisms play in the periodontitis in household pets particularly, dogs in ecologic and therapeutic terms, since these animals might acquire these periodontopahogens from their respective owners.Neste estudo, a presença de patógenos periodontais, Porphyromonas gingivalis, Prevotella intermedia, Tannerella forsythensis, Fusobacterium nucleatum, Dialister pneumosintes, Actinobacillus actinomycetemcomitans, Campylobacter rectus, Eikenella corrodens e Treponema denticola foi determinada por PCR, em amostras subgengivais de 40 cães com (25 e sem (15 doença periodontal. Os produtos amplificados pelo PCR para cada espécie bacteriana mostraram amplicons específicos. Dos 25 cães apresentando doença periodontal, P. gingivalis foi detectado em 16 amostras (64%, C. rectus em 9 (36%, A. actinomycetemcomitans em 6 (24%, P. intermedia em 5 (20%, T. forsythensis em 5 (20

Diagnóstico de febre catarral maligna em bovinos do Uruguai

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Marcela Preliasco

2013-01-01

Full Text Available Foi realizado um estudo retrospectivo de 14 focos de febre catarral maligna (FCM em bovinos, detectados nos anos de 1999-2011, a partir dos arquivos da Seção Anatomia Patológica da Divisão de Laboratórios Veterinários (DILAVE "Miguel C. Rubino" Montevideo. Foram analisados os dados epidemiológicos, apresentação clínica e lesões macroscópicas e histopatológicas. Para a detecção do herpesvírus ovino tipo 2 (OvHV-2 foi utilizada a técnica de PCR sobre as amostras do sistema nervoso central de bovinos de 12 focos. Os surtos ocorreram principalmente nos meses de primavera e verão, na região norte do país. Em 64% (9/14 dos focos ocorreram episódios individuais da enfermidade, enquanto que os casos coletivos foram 5, nos quais a morbidade e mortalidade oscilaram entre 2% e 5%, sendo a letalidade 100% em todos os relatos. Em 50% dos surtos foi confirmado o contato direto entre bovinos e ovinos, enquanto no restante não havia tal informação. Clinicamente predominaram os sinais de opacidade bilateral da córnea, conjuntivite, secreção nasal e ocular mucopurulenta, assim como a síndrome nervosa. Os achados de necropsia mais frequentes foram opacidade bilateral da córnea e lesões inflamatórias nas mucosas. Os achados histopatológicos caracterizaram-se por panvasculite necrótica sistêmica. Foi possível detectar o agente etiológico por PCR em 5 dos 12 casos analisados.

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

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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

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

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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

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)

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)

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.

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

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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.

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

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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

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.

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

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

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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.

Prevalência de marcadores imuno-hematológicos em recém-nascidos ao nascimento e em suas respectivas mães e incidência de doença hemolítica numa maternidade de São Paulo

OpenAIRE

Cianciarullo, Marco Antonio; Ceccon, Maria Esther Jurfest; Vaz, Flávio Adolfo Costa

2003-01-01

A introdução da imunoglobulina anti-D diminuiu a incidência da doença hemolítica neonatal por isoimunização Rh, porém persiste este diagnóstico por outros anticorpos mais raros e o avanço tecnológico tornou possível a detecção destes anticorpos. OBJETIVOS: Verificar a prevalência de marcadores imuno-hematológicos, representados pelos testes de Coombs indireto, direto e de eluição com identificação do anticorpo detectado; incidência de doença hemolítica e de tratamento entre os recém-nascidos ...

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

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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.

Evaluation of resampling applied to UAV imagery for weed detection using OBIA

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Borra, I.; Peña Barragán, José Manuel; Torres Sánchez, Jorge; López Granados, Francisca

2015-01-01

Los vehículos aéreos no tripulados (UAVs) son una tecnología emergente en el estudio de parámetros agrícolas por sus características y por portar sensores en diferente rango espectral. En este trabajo se ha detectado y cartografiado rodales de malas hierbas en fase temprana mediante análisis OBIA para elaborar mapas que optimicen el tratamiento herbicida localizado. Se ha aplicado resampling (resampleo) sobre imágenes tomadas en campo desde un UAV (UAV-I) para crear una nueva imagen con disti...

Tratamentos alternativos no combate à Varroa. Aplicação na apicultura biológica em Trás-os-Montes e Alto Douro

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Vilas-Boas, Miguel

2004-01-01

A varroose é urna doença parasitária externa das abelhas causada por um ácaro denominado por Varroa destructor. Este parasita original mente presente nas abelhas melíferas asiáticas Apis cerana, onde coexiste numa relação simbiótica parasita/hospedeiro,foi detectado pela primeira vez em 1963 em colónias de abelhas Appis mellífera. Este salto imediato não Ihes permitiu criar os seus mecanismos de resistência ao parasita, pelo que se verifica actualmente urna mortalidade eleva...

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

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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.

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

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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

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

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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.

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

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.

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.

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...

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

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.

Prevalencia del protozoario Perkinsus sp. en un cultivo de ostión japonés Crassostrea gigas en Sinaloa, México

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Lizeth Carolina Villanueva-Fonseca

2013-11-01

Full Text Available Crassostrea gigas es un molusco bivalvo de gran importancia comercial. En el noroeste de México su producción es afectada por mortalidades cuyo origen infeccioso no ha sido determinado claramente. En este trabajo se determinó la prevalencia e intensidad de la infección por Perkinsus sp. en un cultivo de C. gigas en el ciclo 2011-2012. El cultivo se hizo en un sistema de línea suspendida con densidades de 28 y 42 ostiones/canasta y se determinó un tamaño de muestra de 30 ostiones por mes. La detección de Perkinsus sp. se hizo de acuerdo a los protocolos de la Organización Mundial de Sanidad Animal (OIE para Medio Fluido de Tioglicolato y PCR. Con ambos métodos se determinó la prevalencia de Perkinsus sp., que varió entre 3,3 y 40%. La intensidad de la infección estuvo en niveles 1 y 2, de acuerdo a la escala de Mackin. La mortalidad acumulativa en las densidades de 28 y 42 ostiones por canasta fue del 4 y 6%, respectivamente. Las mayores mortalidades del ostión y las mayores prevalencias de Perkinsus sp. ocurrieron en septiembre (2,7 y 16,6% y octubre (1,5 y 23,3%, respectivamente, cuando la temperatura fue alta. En conclusión, Perkinsus sp. fue detectado en un cultivo de C. gigas en el estero La Pitahaya con prevalencia moderada, baja intensidad de infección y mayor presencia en los meses más calurosos del ciclo de cultivo.

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)

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.

Reacción en cadena de la polimerasa: una contribución para el diagnóstico de la tuberculosis extrapulmonar y de las micobacteriosis Polymerase chain reaction (PCR: a contribution for diagnosis of extrapulmonary tuberculosis and micobacteriosis

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GLORIA PUERTO

2007-06-01

Full Text Available Introducción. La Organización Mundial de la Salud define la tuberculosis extrapulmonar como la enfermedad tuberculosa localizada en cualquier órgano diferente a los pulmones y, la micobacteriosis, como la infección bacteriana producida por micobacterias no tuberculosas. El diagnóstico de tuberculosis extrapulmonar y micobacteriosis se hace fundamentalmente por el cultivo y la posterior identificación de la micobacteria por pruebas bioquímicas, así como por baciloscopia para el primer caso. El cultivo de micobacterias presenta limitaciones, especialmente, por el tiempo de incubación que puede tardar hasta 12 semanas y por la sensibilidad para determinar la presencia del microorganismo. Actualmente, se utilizan herramientas moleculares que permiten disminuir el tiempo de diagnóstico. Entre ellas está la reacción en cadena de la polimerasa (PCR que es una prueba rápida con buena sensibilidad y especificidad para la identificación de patógenos. Objetivo. Implementar la (PCR como apoyo para el diagnóstico de la tuberculosis extrapulmonar y la micobacteriosis, para disminuir el tiempo en la obtención de resultados y contribuir a la instauración de tratamientos oportunos para estas patologías. Materiales y métodos. El estudio se llevó a cabo entre junio de 2005 y agosto de 2006. Se procesaron 57 muestras clínicas provenientes de 15 centros hospitalarios del país, analizadas por PCR para la secuencia de inserción IS6110 y para el gen hsp65. Se incluyeron datos clínicos del paciente, edad, sexo y prueba de VIH. Resultados. Los resultados de amplificación para PCR IS6110 fueron positivos en 6 pacientes, lo que sugiere tuberculosis extrapulmonar, y para PCR hsp65, en igual número, lo que sugiere un proceso de micobacteriosis. Se confirmó el diagnóstico de tuberculosis meníngea en 4 (6,9% pacientes, seguida por tuberculosis en piel y tuberculosis miliar, cada una en un (1,7% paciente. Se identificaron micobacterias no

OPTIMIZACIÓN DE UN PROTOCOLO DEL AISLAMIENTO DEL ADN Y DE UN SISTEMA DE AMPLIFICACIÓN ISSR-PCR PARA Ceratozamia mexicana Brongn. (Zamiaceae.

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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.

Detection of GBV-C/HGV RNA in cervico-vaginal smears from healthy individuals

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Maria Angelica Ehara Watanabe

2008-10-01

Full Text Available The purpose of the present study was to evaluate the sexual transmission of GBV-C/HGV, through RNA detection in cervicovaginal smears. Therefore the GBV-C/HGV RNA in cervicovaginal smears from apparently healthy women was investigated using routine proceedings for prophylactic screening to cervical cancer. GBV-C/HGV RNA was detected by reverse transcriptase and polymerase chain reaction (RT-PCR. Only one woman presented co-infection with human papilloma virus (HPV. The GBV-C/HGV RNA was detected in 13/73 (17.57% healthy women and it's prevalence in participating women between 28-43 years old was 53.85%. No association was found with GBV-C/HGV for the age of first sexual intercourse and number of pregnancies. In GBV-C/HGV RNA positive women, 69.23% were married. In conclusion, the present findings show that cervical and vaginal specimens could contain the GBV-C/HGV RNA.O objetivo do presente estudo foi avaliar a transmissão sexual de GBV-C/HBV, através da detecção do RNA viral em raspados cérvico-vaginais. Portanto, a presença do RNA GBV-C/HGV foi investigada em raspados cérvico-vaginais em mulheres aparentemente saudáveis que realizaram exames preventivos para câncer cervical. GBV-C/HGV RNA foi detectado por reação de transcriptase reversa e reação em cadeia da polimerase (RT-PCR. Apenas uma mulher apresentou a co-infecção com o papiloma vírus humano (HPV. O RNA GBV-C/HGV foi detectado em 13/73 (17,57% mulheres saudáveis e sua prevalência entre participantes da idade de 28-43 anos foi de 53,85%. Não foi observada relação entre a presença do RNA GBV-C/HGV com a idade de primeira relação sexual, nem com o número de gestações. Entre as mulheres que apresentavam o RNA viral, 69,23% eram casadas. O presente estudo demonstrou que secreções cérvico-vaginais podem conter o RNA viral GBV-C/HBV.

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

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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

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.

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

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.

[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.

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

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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

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

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Helena Lage Ferreira

2009-08-01

Full Text Available 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 established test for the attachment (G gene. All the RT-PCR tested assays were able to detect the AMPV/A. The lower detection limits were observed using the N-, F- based RRT-PCR and F-based conventional RT-PCR (10(0.3 to 10¹ TCID50 mL-1. The present study suggests that the conventional F-based RT-PCR presented similar detection limit when compared to N- and F-based RRT-PCR and they can be successfully used for AMPV/A detection.O metapneumovírus aviário (AMPV pertence ao gênero Metapneumovirus, família Paramyxoviridae. Isolamento viral, sorologia e detecção do RNA genômico são atualmente as técnicas utilizadas para o diagnóstico desse agente. O objetivo do presente estudo foi comparar a detecção de RNA viral de seis isolados de AMPV, subtipo A (AMPV/A, utilizando diferentes métodos de RT-PCR convencional e real time RT-PCR (RRT-PCR. Duas novas técnicas de RT-PCR convencional e duas técnicas de RRT-PCR, ambas para a detecção dos genes da nucleoproteína (N e da proteína de fusão (F, foram comparadas com um RT-PCR previamente estabelecido para a detecção do AMPV (gene da glicoproteína -G. Todos esses métodos foram capazes de detectar os isolados AMPV/A. As técnicas RRT-PCR (genes F e N mostraram os menores limites de detecção (10(0.3 to 10¹ TCID50 mL-1. Os resultados sugerem que as técnicas RT-PCR convencional (gene F e as técnicas de RRT-PCR (gene F e N desenvolvidas no presente estudo podem ser utilizadas com sucesso para a detecção do

Estudo epidemiológico e avaliação de fatores de risco da infecção por Toxoplasma gondii e achados clinico-patológicos da infecção aguda em cães admitidos em um Hospital Escola Veterinário

Directory of Open Access Journals (Sweden)

Angelita D. Strital

Full Text Available RESUMO: Esse trabalho teve como objetivo estudar a prevalência e respectivos fatores de risco para infecção do Toxoplasma gondii em cães provenientes de uma população hospitalar. Além disso, avaliou-se as taxas de ocorrência e as repercussões clínico-patológicas da infecção aguda pelo T. gondii nesses animais. Anticorpos foram detectados em 7% (26/386 da população estudada, composta de 386 cães de ambos os sexos e diferentes raças e idades. Somente as variáveis, ingestão de vísceras, origem rural e contato com bovinos apresentaram valores significativos com p<0.05. Adicionalmente os cães de origem rural apresentaram maiores risco (OD=7.00 de infecção do que aqueles de origem urbana. Em 6,5% (25/386 foram detectados títulos de contato (entre 16 e 256; esses títulos não significam necessariamente infecção aguda e sim apenas exposição prévia. É de fundamental importância o reconhecimento da infecção prévia por T. gondii nesses pacientes hospitalares. Dependendo da causa da admissão, mesmo não sendo a toxoplasmose a responsável, o paciente deve receber o tratamento anti-protozoário profilaticamente ou ser monitorado para posterior tratamento em caso de reagudização da enfermidade por recrudescência dos bradizoítos encistados. Apenas um animal (3.44%, 1/386 foi admitido com titulação elevada, o qual pode ser sugestivo de infecção aguda (titulo de 4096. Embora o animal com infecção aguda tenha sido apresentado com sinais neurológicos, cautela é necessária para não extrapolar uma falsa interpretação que a toxoplasmose é a grande responsável por quadros neurológico, uma vez que inúmeros outros casos incluídos nesse estudo tinham manifestações neurológicas e não tinham títulos de infecção aguda, nem mesmo título de contato prévio. A toxoplasmose aguda não foi uma afecção clínica expressiva nessa ambiência hospitalar, no entanto diagnóstico diferencial deve ser feito nos pacientes

Caracterização fenogenotípica da resistência antimicrobiana em Staphylococcus spp. isolados de mastite bovina

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Elaine C.L. Mendonça

2012-09-01

Full Text Available A utilização de antibióticos no controle das infecções intramamárias e na eliminação de prováveis fontes de infecção nas fazendas leiteiras se constitui em importante medida de controle. No entanto, o uso inadequado de antibióticos no tratamento da doença pode selecionar cepas resistentes e comprometer a eficiência do tratamento. Bactérias do gênero Staphylococcus spp. estão entre os principais agentes etiológicos da mastite bovina e são freqüentemente resistentes aos antimicrobianos, em especial aos beta-lactâmicos, principalmente por dois mecanismos distintos: a produção da enzima extracelular beta-lactamase, codificada pelo gene blaZ, e a produção de PBP2a ou PBP2´, uma proteína ligante de penicilina de baixa afinidade, codificada pelo gene mecA. A expressão do gene mecA é constitutiva ou induzida por antibióticos betalactâmicos, como a oxacilina e cefoxitina. O gene mecA está inserido no cromossomo através de um elemento genético móvel, denominado cassete estafilocócico cromossômico mec (SCCmec. O presente estudo avaliou o perfil fenogenotípico de resistência aos beta-lactâmicos em 250 isolados de Staphylococcus spp., utilizando os marcadores oxacilina e cefoxitina, de modo a produzir dados que possam contribuir para o conhecimento da resistência antimicrobiana em algumas propriedades leiteiras das regiões Sul-Fluminense e Metropolitana do Estado do Rio de Janeiro com o objetivo de subsidiar a implementação de medidas de controle dessa enfermidade. A avaliação da resistência foi feita a partir de 8 diferentes testes fenotípicos, sendo obtidos 54 perfis. Os testes de difusão em disco simples e ágar screen com oxacilina foram utilizados como "padrão ouro" para os cálculos dos valores de sensibilidade, especificidade e predição por serem preconizados pelo CLSI veterinário. O teste de difusão em disco simples com cefoxitina foi o de melhor desempenho na predição da resistência a

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...

Caracterización y significado de las rocas silíceas y ferruginosas del Paleoceno de Zamora

OpenAIRE

Bustillo, Mª Ángeles; Martín Serrano, Ángel

1980-01-01

Se estudian las rocas silíceas y ferruginosas del Paleógeno de la provincia de Zamora dentro del contexto general de la sedimentación fluvial. El análisis detallado de las muestras es realizado por técnicas petrográficas en lámina delgada y probeta pulida, determinando además su composición mineralógica por difracción de Rayos X. Los minerales silíceos detectados son ópalo C y ópalo C-T mientras que como minerales de hierro aparecen hidróxidos amorfos, hematites y goetita. Las conclusiones...

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

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 ...

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.

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.

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.

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

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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.

Rickettsial spotted fever in capoeirão Village, Itabira, Minas Gerais, Brazil Rickettsiose do grupo da febre maculosa na Vila de Capoeirão, Itabira, Minas Gerais, Brasil

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Manoella Campostrini Barreto Vianna

2008-10-01

Full Text Available The present study investigated the infection by spotted fever rickettsia in an endemic area for Brazilian spotted fever (BSF; caused by Rickettsia rickettsii in Minas Gerais State, Brazil. Human, canine and equine sera samples, and Amblyomma cajennense adult ticks collected in a rural area of Itabira City, Minas Gerais State were tested for rickettsial infection. Through Immunofluorescence Assay (IFA we demonstrated the presence of antibodies anti-R. rickettsii in 8.2%, 81.3% and 100% of the human, canine and equine sera, respectively. None of the 356 tick specimens analyzed were positive for Rickettsia by the hemolymph test or Polymerase Chain Reaction technique (PCR for the htrA and the gltA genes. Our serological results on horses and dogs (sentinels for BSF appoint for the circulation of a SFG Rickettsia in the study area, however in a very low infection rate among the A. cajennense tick population.O presente estudo investigou a infecção por rickéttsias do grupo da febre maculosa (GFM em área endêmica para febre maculosa brasileira (FMB; causada por Rickettsia rickettsii no Estado de Minas Gerais, Brasil. Amostras de soros de humanos, cães e eqüídeos, e carrapatos Amblyomma cajennense adultos colhidos em um povoado rural em Itabira, Minas Gerais foram testados para infecção por Rickettsia. Pela Reação de Imunofluorescência Indireta (RIFI foram detectados anticorpos anti-R. rickettsii em 8,2% dos soros humanos, 81,3% dos cães e em 100% dos eqüídeos. Nenhum dos 356 carrapatos se mostrou positivo para Rickettsia no teste de hemolinfa e na reação em cadeia pela polimerase (PCR objetivando amplificar fragmentos de DNA dos genes htrA and the gltA. Os resultados sorológicos em eqüinos e cães (sentinelas para FMB apontam para a circulação de uma rickéttsia do GFM na área do estudo, porém, numa freqüência de infecção muito baixa na população do carrapato A. cajennense.

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.

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.

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...

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.

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.

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

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

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...

Análise espaço-temporal da clorose variegada dos citros no Noroeste do Paraná, com uso de PCR para detecção de Xylella fastidiosa = Spatio-temporal analysis of the citrus variegated chlorosis (CVC in the Northwest of Paraná, using PCR for detection of Xylella fastidiosa

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William Mário de Carvalho Nunes

2006-07-01

Full Text Available A citricultura é afetada por inúmeras doenças, como a clorose variegada do citros (CVC, causada pela bactéria Xylella fastidiosa. O objetivo deste trabalho foi determinar a distribuição espacial da doença dentro de pomares comerciais do Noroeste do Paraná com o uso de métodos moleculares. Foram selecionados pomares sintomáticos para CVC com as variedades ‘Pêra’, ‘Valência’ e ‘Folha Murcha’ (Citrus sinensis Osbeck. Foram marcadas para cada variedade, 4 plantas-referência positivas para CVC (por sintomas e análise molecular e 8 plantas ao redor de cada uma das plantas-referência foram amostradas, num total de 36 plantas por variedade. Realizou-se o teste da Reação da Polimerase em Cadeia (PCR para detecção da bactéria e na mesma época foram conduzidas avaliações visuais de sintomas de CVC. Os resultados da análise temporal, utilizando-se os modelos Monomolecular, Logístico e Gompertz, apontaram o modelo Logístico como o que melhor se ajustou para descrever o comportamento da doença no tempo, para todas as variedades estudadas. Observou-se que o comportamento espacial da doença diferiu quando a mesma área foiavaliada pelos métodos visual e molecular, resultando em uma diferença no padrão espacial das áreas avaliadas. Portanto, ambos os métodos empregados, sintomas e PCR, foram capazes de constatar asmudanças no padrão espacial apresentado, sendo que a análise molecular (PCR foi mais sensível para detectar as mudanças ocorridas.Countless diseases affect the citriculture, as the citrus variegated chlorosis (CVC which is caused by the bacteria Xylella fastidiosa.The aim of this work was to determine the space distribution of the disease inside commercial orchards in the Northwest of Paraná, using molecular methods. Symptomatic orchards were selected for CVC with the varieties 'Pêra', 'Valência' and 'Folha Murcha' (Citrus sinensis Osbeck. For each variety, 4 positive reference-plant for CVC

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)

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

Transgene detection by digital droplet PCR.

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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.

Expression of human bone morphogenetic protein (BMP-2 and BMP-4 genes in transgenic bovine fibroblasts Expressão dos genes bone morphogenetic protein (BMP-2 e BMP-4 em fibroblastos bovinos transgênicos

Directory of Open Access Journals (Sweden)

C. Oleskovicz

2004-08-01

Full Text Available cDNAs dos genes bone morphogenetic protein-2 (BMP-2 e bone morphogenetic protein-4 (BMP-4 foram sintetizados a partir de RNA total extraído de tecidos ósseos de pacientes que apresentavam trauma facial (fraturas do maxilar entre o 7º e o 10º dia pós-trauma e clonados num vetor para expressão em células mamíferas, sob controle do promotor de citomegalovírus (CMV. Os vetores contendo os genes BMP-2 e o BMP-4 foram utilizados para a transfecção de fibroblastos bovinos. mRNAs foram indiretamente detectados por RT-PCR nas células transfectadas. As proteínas BMP-2 e BMP-4 foram detectadas mediante análises de Western blot. Os resultados demonstram a possibilidade de produção desses fatores de crescimento celular em fibroblastos bovinos. Essas células poderão ser utilizadas como fontes doadoras de material genético para a técnica de transferência nuclear na geração de animais transgênicos.

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.

[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

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.

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...

Comparación de los métodos de inmunoensayo enzimático automatizado (VIDAS y PCR para la detección de Salmonella spp. en expendios de la ciudad de Santa Marta (Colombia

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Libardo Acosta

2013-01-01

Full Text Available Objetivo: comparar los métodos de inmunoensayo enzimático automatizado VIDAS, (Vi- tek Immunodiagnostic Assay System, y el análisis molecular por Reacción en Cadena de la Polimerasa (PCR para detectar Salmonella spp en expendios de la ciudad de Santa Marta (Colombia. Materiales y métodos: Previamente se reportó la estandarización de la técnica de PCR para la detección de Salmonella spp. en un lapso de 12 horas. En colaboración con el Labo- ratorio de Salud Pública del departamento del Magdalena se procedió a tomar un total de 65 muestras de alimentos para análisis de vigilancia rutinaria, discriminados así: carne de res: 14 (21.5 %, embutidos: 18 (27.7 %, pollo: 7(10.8 %, pescado: 3(4.6 %, harinas: 13 (20 %, lácteos: 5 (7.7 % salsas: 4(6.2 % y ensalada: 1(1.5 %, en la ciudad de Santa Marta entre septiembre y noviembre de 2010. Las 65 muestras fueron sometidas a análisis microbiológico para la determinación de Salmonella spp. en el Laboratorio de Salud Pú- blica del departamento del Magdalena siguiendo el protocolo del VIDAS. Una alícuota de 1 mL del preenriquecimiento no selectivo fue enviada refrigerada al laboratorio de biología molecular de la Universidad Cooperativa de Colombia para ser analizada por PCR. Resultados: Los resultados muestran que los alimentos analizados en el Laboratorio de Salud Pública del departamento del Magdalena con VIDAS presentaron Salmonella spp. únicamente en cárnicos: 5/65 (7.7 %, mientras que esas mismas muestras analizadas por PCR mostraban la presencia de Salmonella spp. en 36/65 (55.4 %. Conclusiones: Los resultados indican que la PCR puede ser aplicada para realizar vigilan- cia epidemiológica y obtener resultados cualitativos confiables en menor tiempo.

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...

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

Uso de la técnica SSCP para detectar mutaciones puntuales del ADN mitocondrial humano

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Alejandro Estrada-Cuzcano

2013-05-01

Full Text Available En el presente trabajo se evalúa la técnica de SSCP (polimorfismo de conformación de cadena individual de ADN para detectar mutaciones puntuales, tanto por su sensibilidad en la detección (alrededor 80% en condiciones ideales, como por su implementación fácil y económica. Se utilizaron como controles positivos y negativos, ADN de voluntarios caracterizados previamente para las mutaciones puntuales de 5 RFLPs mitocondriales. Para la optimización de la prueba fueron ensayadas concentraciones variables del tampón TBE (1X y 0,5X y del glicerol (10%, 5% y 0 en geles de poliacrilamida. Cuatro de los 5 RFLPs fueron detectados en las condiciones utilizadas y pueden ser usados en estudios de rutina sin usar enzimas de restricción. Además, la técnica SSCP permitió determinar mutaciones desconocidas en un segmento de 394 nucleótidos de la región hipervariable (HVI del ADNmt. Diferencias en correspondencia a los distintos haplotipos fueron detectados e incluso permitió discernir grupos dentro del mismo subtipo. La secuenciación de dos muestras del subtipo B1 con migración diferencial en SSCP, corroboró la existencia de siete nucleótidos distintos

Estrategia de genotipado del gen FMR1: Método de diagnóstico alternativo para el Síndrome X Frágil y otras enfermedades por expansión de trinucleotidos

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Saúl Lindo-Samanamud

2013-10-01

Full Text Available Objetivos: Diseñar una estrategia alternativa por PCR para el genotipado de secuencias ricas en citosinas, basada en modificación nucleotídica. Material y métodos: Se modificó el gen FMR1 nativo de ocho individuos clínicamente no afectados por el Síndrome X frágil, cambiando las citosinas por uracilos, empleando bisulfito de sodio. El ADN modificado fue purificado y cuantificado por espectrofotometría. Las estructuras alternativas y potenciales islas CpG que adopta el microsatélite inestable fueron simuladas con los programas MFOLD y CpGplot. Se generaron cebadores específicos que hibriden tanto con el microsatélite modificado (Primer T y con una secuencia modificada de las islas CpG (Primer M, utilizando el programa MethPrimer. Finalmente, ambas secuencias fueron amplificadas por PCR y los amplicones fueron separados por electroforesis en gel de poliacrilamida (PAGE por sus siglas en inglés al 6% y visualizados con tinción de nitrato de plata. Resultados: La modificación del ADN fue evidenciada por espectrofotometría al uracilo. Las estructuras observadas en la simulación fueron las horquillas encontrándose dos potenciales islas CpG. La amplificación con los cebadores T, confirmó el diseño in silico desarrollado para abordar la estructura en horquillas. La amplificación con los cebadores M permitió detectar metilación de la primera isla CpG del gen FMR1.Conclusión: Se propone un diseño alternativo para amplificación de secuencias de microsatélite que contengan citosinas metiladas y no metiladas. Se requieren estudios posteriores con muestras de ADN que contengan microsatélites muy expandidos para validar su aplicación para diagnóstico molecular.

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

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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.

Development of Nested PCR, Multiplex PCR, and Loop-Mediated Isothermal Amplification Assays for Rapid Detection of Cylindrocladium scoparium on Eucalyptus

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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.

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

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.

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...

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.

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

RT-PCR Protocols - Methods in Molecular Biology

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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...........

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

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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.

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...

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.

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...

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...

Infecção natural por hemoparasitos em bezerros submetidos à quimio-profilaxia aos 30 dias de idade Natural infection by hemoparasites in calves submitted to chemo -prophylaxis wonder 30 days of age

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Rosângela A. Da Silva

2007-09-01

Full Text Available O complexo Tristeza Parasitária acarreta grandes prejuízos à pecuária bovina nacional. Os principais agentes etiológicos são Babesia bigemina, B. bovis e Anaplasma marginale, sendo o carrapato Boophilus microplus o principal vetor. Este trabalho relata a ocorrência de infecção natural por hemoparasitos da tristeza parasitária bovina em 36 bezerros com alta infestação natural por carrapatos e submetidos à quimioprofilaxia aos 30 dias de idade. Babesia bigemina (33,3%, B. bovis (11,1% e A. marginale (13,9% foram detectados em esfregaços sangüíneos de 16 animais (44,4% de diferentes idades. Seis bezerros apresentaram sintomas (16,7% e um morreu (2,8%. O número de casos clínicos foi decorrente de uma associação de fatores, destacando-se a alta infestação precoce por carrapatos e a baixa imunidade passiva em período em que os bezerros ainda não haviam desenvolvido imunidade ativa suficiente.The tick-borne disease (TBD brings great damages to cattle breeding. The most important etiologic agents are Babesia bigemina, B. bovis and Anaplasma marginale, being the tick Boophilus microplus the main vector. This work reports the occurrence of natural infection by hemoparasites of TBD in 36 calves with high ticks natural infestation submitted to chemoprophylaxis with 30 days year-old. The blood smears from animals of different ages were analized and were found B. bigemina (33.3%, B. bovis (11.1% and A. marginale (13.9%. Six animals had clinical symptoms (16.7% and one dead (2.8%. The number of clinical cases ocurred in consequence of an association of factors as high infestation of ticks and low passive immunity in period that calves had not developed enough active immunity.

Pitfalls in PCR troubleshooting: Expect the unexpected?

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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.

Uso de álcool em vítimas de acidentes de trânsito: estudo do nível de alcoolemia Uso de alcohol en accidentes de tránsito: estudio del nivel de alcoholemia Alcohol use and traffic accidents: a study of alcohol levels

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Ângela Maria Mendes Abreu

2010-06-01

Full Text Available Trata-se de estudo exploratório descritivo. O objetivo foi correlacionar os níveis de alcoolemia, detectados nas vítimas fatais por acidentes de trânsito, na cidade do Rio de Janeiro, a partir dos registros do Instituto Medico Legal -IML, com o perfil da vítima e as características dos acidentes. Os dados foram levantados no arquivo do IML, por meio dos prontuários de vítimas fatais por acidentes de trânsito, compilados e tabulados Poe meio do programa estatístico SPSS, no período compreendido entre janeiro e maio de 2005. Avaliaram-se 348 prontuários de vítimas fatais por acidentes de trânsito. Desses, apenas 94 realizaram o exame de alcoolemia, sendo que 83 apresentaram alcoolemia positiva e 60,2% níveis acima de 0,6g/l. Evidenciou-se o envolvimento do álcool com vítimas fatais nos acidentes de trânsito em níveis acima e abaixo de 0,6g/l de álcool por litro de sangue.Se trata de estudio epidemiológico exploratorio y descriptivo. El objetivo fue correlacionar los niveles de alcoholemia detectados en las víctimas fatales por accidentes de tránsito, en la ciudad de Río de Janeiro (datos de los registros del Instituto Médico Legal/IML con el perfil de la víctima y las características de los accidentes. Los datos fueron recolectados del archivo del IML, a través de los registros que constaban en las fichas de víctimas fatales por accidentes de tránsito; fueron compilados y se elaboraron tablas con el Programa estadístico SPSS, en el período comprendido entre enero y mayo de 2005. Fueron levantados datos sobre 348 víctimas fatales por accidentes de tránsito, de estos, apenas 94 realizaron el examen de alcoholemia, siendo que en 83 fueron detectados niveles de alcoholemia; el 60,2% presentó niveles por encima de 0,6g de alcohol por litro de sangre. Se mostró evidente el envolvimiento del alcohol en accidentes de tránsito con víctimas fatales, en niveles por encima y por debajo de 0,6g de alcohol por litro de

Nuevas moléculas asociadas al cáncer de ovario para la mejora de su diagnóstico y tratamiento

OpenAIRE

Lanau Angulo, Lucia

2017-01-01

El cáncer de ovario (CO) es el cáncer ginecológico más letal y la quinta causa de muerte por cáncer en mujeres del mundo occidental. Debido a la inespecificidad de los síntomas y a la falta de técnicas diagnósticas eficaces, el CO es detectado en el 85% de los casos en estadio avanzado, resultando en una tasa de supervivencia a 5 años del 28%. En contraste, la tasa aumenta hasta el 92% si se detecta en estadios iniciales. Por lo tanto, existe una necesidad clínica evidente de identificar nuev...

IMPLEMENTACIÓN DE LA PCR (REACCIÓN EN CADENA DE LA POLIMERASA PARA EL DIAGNÓSTICO DE LA BRUCELOSIS DE BOVINOS EN EL ECUADOR

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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.

Performance of Droplet Digital PCR in Non-Invasive Fetal RHD Genotyping - Comparison with a Routine Real-Time PCR Based Approach.

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Iveta Svobodová

Full Text Available Detection and characterization of circulating cell-free fetal DNA (cffDNA from maternal circulation requires an extremely sensitive and precise method due to very low cffDNA concentration. In our study, droplet digital PCR (ddPCR was implemented for fetal RHD genotyping from maternal plasma to compare this new quantification alternative with real-time PCR (qPCR as a golden standard for quantitative analysis of cffDNA. In the first stage of study, a DNA quantification standard was used. Clinical samples, including 10 non-pregnant and 35 pregnant women, were analyzed as a next step. Both methods' performance parameters-standard curve linearity, detection limit and measurement precision-were evaluated. ddPCR in comparison with qPCR has demonstrated sufficient sensitivity for analysing of cffDNA and determination of fetal RhD status from maternal circulation, results of both methods strongly correlated. Despite the more demanding workflow, ddPCR was found to be slightly more precise technology, as evaluated using quantitative standard. Regarding the clinical samples, the precision of both methods equalized with decreasing concentrations of tested DNA samples. In case of cffDNA with very low concentrations, variance parameters of both techniques were comparable. Detected levels of fetal cfDNA in maternal plasma were slightly higher than expected and correlated significantly with gestational age as measured by both methods (ddPCR r = 0.459; qPCR r = 0.438.

Progreso en el logro de los Objetivos de Desarrollo del Milenio: la mortalidad por cáncer de cérvix desciende en Colombia

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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.

Detection of five potentially periodontal pathogenic bacteria in peri-implant disease: A comparison of PCR and real-time PCR.

Science.gov (United States)

Schmalz, Gerhard; Tsigaras, Sandra; Rinke, Sven; Kottmann, Tanja; Haak, Rainer; Ziebolz, Dirk

2016-07-01

The aim of this study was to compare the microbial analysis methods of polymerase chain reaction (PCR) and real-time PCR (RT-PCR) in terms of detection of five selected potentially periodontal pathogenic bacteria in peri-implant disease. Therefore 45 samples of healthy, mucositis and peri-implantitis (n = 15 each) were assessed according to presence of the following bacteria using PCR (DNA-strip technology) and RT-PCR (fluorescent dye SYBR green-system): Aggregatibacter actinomycetemcomitans (Aa), Porphyromonas gingivalis (Pg), Treponema denticola (Td), Tanerella forsythia (Tf), and Fusobacterium nucleatum (Fn). There were no significant correlations between the bacterial and disease patterns, so the benefit of using microbiological tests for the diagnosis of peri-implant diseases is questionable. Correlations between the methods were highest for Tf (Kendall's Tau: 0.65, Spearman: 0.78), Fn (0.49, 0.61) and Td (0.49, 0.59). For Aa (0.38, 0.42) and Pg (0.04, 0.04), lower correlation values were detected. Accordingly, conventional semi-quantitative PCR seems to be sufficient for analyzing potentially periodontal pathogenic bacterial species. Copyright © 2016 Elsevier Inc. All rights reserved.

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....

Development and Evaluation of a PCR and Mass Spectroscopy-based (PCR-MS) Method for Quantitative, Type-specific Detection of Human Papillomavirus

Science.gov (United States)

Patel, Divya A.; Shih, Yang-Jen; Newton, Duane W.; Michael, Claire W.; Oeth, Paul A.; Kane, Michael D.; Opipari, Anthony W.; Ruffin, Mack T.; Kalikin, Linda M.; Kurnit, David M.

2010-01-01

Knowledge of the central role of high-risk human papillomavirus (HPV) in cervical carcinogenesis, coupled with an emerging need to monitor the efficacy of newly introduced HPV vaccines, warrant development and evaluation of type-specific, quantitative HPV detection methods. In the present study, a prototype PCR and mass spectroscopy (PCR-MS)-based method to detect and quantitate 13 high-risk HPV types is compared to the Hybrid Capture 2 High Risk HPV DNA test (HC2; Digene Corp., Gaithersburg, MD) in 199 cervical scraping samples and to DNA sequencing in 77 cervical tumor samples. High-risk HPV types were detected in 76/77 (98.7%) cervical tumor samples by PCR-MS. Degenerate and type-specific sequencing confirmed the types detected by PCR-MS. In 199 cervical scraping samples, all 13 HPV types were detected by PCR-MS. Eighteen (14.5%) of 124 cervical scraping samples that were positive for high-risk HPV by HC2 were negative by PCR-MS. In all these cases, degenerate DNA sequencing failed to detect any of the 13 high-risk HPV types. Nearly half (46.7%) of the 75 cervical scraping samples that were negative for high-risk HPV by the HC2 assay were positive by PCR-MS. Type-specific sequencing in a subset of these samples confirmed the HPV type detected by PCR-MS. Quantitative PCR-MS results demonstrated that 11/75 (14.7%) samples contained as much HPV copies/cell as HC2-positive samples. These findings suggest that this prototype PCR-MS assay performs at least as well as HC2 for HPV detection, while offering the additional, unique advantages of type-specific identification and quantitation. Further validation work is underway to define clinically meaningful HPV detection thresholds and to evaluate the potential clinical application of future generations of the PCR-MS assay. PMID:19410602

Development and evaluation of a PCR and mass spectroscopy (PCR-MS)-based method for quantitative, type-specific detection of human papillomavirus.

Science.gov (United States)

Patel, Divya A; Shih, Yang-Jen; Newton, Duane W; Michael, Claire W; Oeth, Paul A; Kane, Michael D; Opipari, Anthony W; Ruffin, Mack T; Kalikin, Linda M; Kurnit, David M

2009-09-01

Knowledge of the central role of high-risk human papillomavirus (HPV) in cervical carcinogenesis, coupled with an emerging need to monitor the efficacy of newly introduced HPV vaccines, warrant development and evaluation of type-specific, quantitative HPV detection methods. In the present study, a prototype PCR and mass spectroscopy (PCR-MS)-based method to detect and quantitate 13 high-risk HPV types is compared to the Hybrid Capture 2 High-Risk HPV DNA test (HC2; Digene Corp., Gaithersburg, MD) in 199 cervical scraping samples and to DNA sequencing in 77 cervical tumor samples. High-risk HPV types were detected in 76/77 (98.7%) cervical tumor samples by PCR-MS. Degenerate and type-specific sequencing confirmed the types detected by PCR-MS. In 199 cervical scraping samples, all 13 HPV types were detected by PCR-MS. Eighteen (14.5%) of 124 cervical scraping samples that were positive for high-risk HPV by HC2 were negative by PCR-MS. In all these cases, degenerate DNA sequencing failed to detect any of the 13 high-risk HPV types. Nearly half (46.7%) of the 75 cervical scraping samples that were negative for high-risk HPV by the HC2 assay were positive by PCR-MS. Type-specific sequencing in a subset of these samples confirmed the HPV type detected by PCR-MS. Quantitative PCR-MS results demonstrated that 11/75 (14.7%) samples contained as much HPV copies/cell as HC2-positive samples. These findings suggest that this prototype PCR-MS assay performs at least as well as HC2 for HPV detection, while offering the additional, unique advantages of type-specific identification and quantitation. Further validation work is underway to define clinically meaningful HPV detection thresholds and to evaluate the potential clinical application of future generations of the PCR-MS assay.

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

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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.

Application of real-time PCR (qPCR) for characterization of microbial populations and type of milk in dairy food products.

Science.gov (United States)

Agrimonti, Caterina; Bottari, Benedetta; Sardaro, Maria Luisa Savo; Marmiroli, Nelson

2017-09-08

Dairy foods represent an important sector of the food market for their nutritional qualities and their organoleptic characteristics, which are often linked to tradition and to region. These products are typically protected by labels such as PDO (Protected Designation of Origin) and PGI (Protected Geographical Indication). Real-time PCR (qPCR) is a fundamental tool in "Food Genomics;" a discipline concerned with the residual DNA in food, which, alongside traditional physical and chemical methods, is frequently used to determine product safety, quality and authenticity. Compared to conventional or "end-point" PCR, qPCR incorporates continuous monitoring of reaction progress, thereby enabling quantification of target DNA. This review describes qPCR applications to the analysis of microbiota, and to the identification of the animal species source of milk from which dairy products have been made. These are important aspects for ensuring safety and authenticity. The various applications of qPCR are discussed, as well as advantages and disadvantages in comparison with other analytical methods.

Polymerase chain reaction methods (PCR in agrobiotechnology

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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.

[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.

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

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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

Splinkerette PCR for mapping transposable elements in Drosophila.

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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.

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

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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.

IDENTIFIKASI TIPE HLA KELAS II DENGAN TEKNIK PCR

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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

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.

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...

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

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.

Detección molecular de potyvirus en hojas y minibulbillos de ajo, Allium sativum, asociados a un programa de producción de semilla limpia

Directory of Open Access Journals (Sweden)

M. Parra Fuentes

2014-07-01

Full Text Available Título en español: Detección molecular de potyvirus en hojas y minibulbillos de ajo, Allium sativum, asociados a un programa de producción de semilla limpia Título en ingles: Molecular detection of potyvirus in leaves and small bulbs of garlic, Allium sativum, associated a clean seed production program Titulo corto: Detección de potyvirus en plantas de ajo Resumen: El ajo (Allium sativum L se reproduce vegetativamente utilizando bulbillos, condición que favorece la propagación de enfermedades, especialmente bacterias, hongos y virus que afectan la calidad y el rendimiento del cultivo. Por este motivo se implementó la identificación molecular por RT-PCR de los potyvirus LYSV y OYDV en el sistema de producción de semilla limpia de ajo en tres clones nacionales. En la fase de producción de semilla limpia mediante micropropagación, se estandarizó el establecimiento de meristemos de ajo. La presencia de potyvirus se analizó en 586 plántulas mediante ELISA y en 70 por RT-PCR. Para la RT-PCR se extrajo ARN a partir de microbulbillos y hojas de plántulas, obteniéndose 1.7 a 226 ng/ml de ARN y se sintetizó entre 35 a 50 ng de cADN. Los resultados obtenidos mostraron que el protocolo de desinfección produjo una viabilidad del 73.6%. El análisis ELISA presentó un saneamiento del 96.1% de las plántulas a potyvirus, mientras que con RT-PCR se identificó la presencia de LYSV en el 8.6% de las muestras evaluadas. El virus del enanismo amarrillo de la cebolla (OYDV no fue detectado en ninguna de las muestras. Los resultados muestran que el cultivo in vitro de meristemos de ajo es una excelente alternativa para la producción de semilla, mostrando un 92% de eficiencia. Además, validan el diagnóstico eficiente del potyvirus LYSV en hojas y microbulbillos de ajo. Palabras clave: Allium sativum, ELISA, micropropagación, LSYV, potyvirus, OYDV, RT-PCR. Abstract:  Garlic (Allium sativum L, reproduces vegetatively using bulbils, condition

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.

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.

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.

Convergencia estratégica en la industria española de fondos de inversión

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Luis Ferruz

2008-01-01

Full Text Available Por medio de la medida de Lakonishok et al. (1992, el artículo aporta evidencia empírica de la convergencia en la asignación estratégica de activos realizada por los fondos españoles de renta variable nacional desde julio de 1997 a junio de 2002. Dicho fenómeno es detectado con mayor intensidad al considerar variaciones estratégicas de mayor relevancia.El estudio también pone de manifiesto una convergencia estratégica intertemporal realmente fuerte entre los fondos gestionados por las entidades financieras españolas más grandes, poniendo de manifiesto que el tamaño de la sociedad gestora es un mecanismo significativo a la hora de explicar dicho fenómeno.

ATPasa de calcio en el sistema nervioso

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Myriam L. Velandia

2001-03-01

Full Text Available La bomba de calcio es una proteína integral de la membrana que regula el calcio libre citoplasmático en concentraciones menores de 0,1mM. En la mayoría de las células eucarióticas está ubicada en la membrana, en el plasmalema o en organelos como el retículo sarcoendoplásmico y los calciosomas. Su actividad está dada por la hidrólisis del ATP, la concentración del ion en el citoplasma y por otros factores que la regulan como la calmodulina, los fosfolípidos y las proteinas-cinasas. Por diferentes métodos, se ha detectado la ATPasa de calcio y su actividad tanto en tejido nervioso central y periférico, como en otros tejidos.

Methylation-Specific PCR Unraveled

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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.

Detection, quantification and genetic variability of Mycoplasma hyopneumoniae from apparently healthy and pneumonic swine

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Yaima Burgher Pulgarón

2015-12-01

Full Text Available As diferenças moleculares entre as estirpes de Mycoplasma hyopneumoniae presentes em pulmões de suínos com pneumonia tem sido estudadas. Porém, estudos comparativos relativos as estirpes presentes nos suínos aparentemente saudáveis não foram levados a cabo. O objetivo do estudo foi a detecção, quantificação e analise molecular de M. hyopneumoniae nos pulmões suínos com e sem lesões pneumônicas. Para a detecção de M. hyopneumoniae usaramse o PCR Multiplo (YAMAGUTI, 2008, o PCR a Tempo Real (STRAIT et al., 2008 e a amplificação de múltiplo VNTR (VRANCKX et al., 2011. A caracterização molecular das estirpes foi realizada mediante a análise do número de copias de VNTR em P97R1, P146R3, H2R1 e H4. O M. hyopneumoniae foi detectado em amostras de suínos saudáveis e pneumônicos e a quantidade de M. hyopneumoniae nas amostras positivas variou com o tipo de ensaio. O maior número de amostras positivas foi identificado pela amplificação de múltiplas VNTR combinado com a eletroforese de capilares. Usando o PCR a Tempo Real, 4.9*104 copias de genoma/mL de M. hyopneumoniae foram detectadas em pulmões aparentemente saudáveis. Uma quantidade média de 3.9*106 copias de genoma/mL de M. hyopneumoniae foi detectada em pulmões pneumônicos. A análise do número de copias de VNTR demonstrou uma elevada variabilidade.

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

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...

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.

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.

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.

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

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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

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.

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.

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.

Have you tried spermine? A rapid and cost-effective method to eliminate dextran sodium sulfate inhibition of PCR and RT-PCR.

Science.gov (United States)

Krych, Łukasz; Kot, Witold; Bendtsen, Katja M B; Hansen, Axel K; Vogensen, Finn K; Nielsen, Dennis S

2018-01-01

The Dextran Sulfate Sodium (DSS) induced colitis mouse model is commonly used to investigate human inflammatory bowel disease (IBD). Nucleic acid extracts originating from these animals are often contaminated with DSS, which is a strong inhibitor of many enzymatic based molecular biology reactions including PCR and reverse-transcription (RT). Methods for removing DSS from nucleic acids extracts exist for RNA, but no effective protocol for DNA or cDNA is currently available. However, spermine has previously been shown to be an effective agent for counteracting DSS inhibition of polynucleotide kinase, which led to the hypothesis, that spermine could be used to counteract DSS inhibition of PCR and RT. We investigated the means of adding spermine in an adequate concentration to PCR based protocols (including qPCR, two-step RT-qPCR, and amplicon sequencing library preparation) to remove DSS inhibition. Within the range up to 0.01g/L, spermine can be added to PCR/qPCR or RT prophylactically without a significant reduction of reaction efficiency. Addition of spermine at the concentration of 0.08g/L can be used to recover qualitative PCR signal inhibited by DSS in concentrations up to 0.32g/L. For optimal quantitative analysis, the concentration of spermine requires fine adjustment. Hence, we present here a simple fluorometric based method for adjusting the concentration of spermine ensuring an optimal efficiency of the reaction exposed to an unknown concentration of DSS. In conclusion, we demonstrate a cost effective and easy method to counteract DSS inhibition in PCR and two-step RT-qPCR. Fixed or fine-tuned concentrations of spermine can be administered depending on the qualitative or quantitative character of the analysis. Copyright © 2017 Elsevier B.V. All rights reserved.

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 ...

The diagnosis of microorganism involved in infective endocarditis (IE by polymerase chain reaction (PCR and real-time PCR: A systematic review

Directory of Open Access Journals (Sweden)

Reza Faraji

2018-02-01

Full Text Available Broad-range bacterial rDNA polymerase chain reaction (PCR followed by sequencing may be identified as the etiology of infective endocarditis (IE from surgically removed valve tissue; therefore, we reviewed the value of molecular testing in identifying organisms' DNA in the studies conducted until 2016. We searched Google Scholar, Scopus, ScienceDirect, Cochrane, PubMed, and Medline electronic databases without any time limitations up to December 2016 for English studies reporting microorganisms involved in infective endocarditis microbiology using PCR and real-time PCR. Most studies were prospective. Eleven out of 12 studies used valve tissue samples and blood cultures while only 1 study used whole blood. Also, 10 studies used the molecular method of PCR while 2 studies used real-time PCR. Most studies used 16S rDNA gene as the target gene. The bacteria were identified as the most common microorganisms involved in infective endocarditis. Streptococcus spp. and Staphylococcus spp. were, by far, the most predominant bacteria detected. In all studies, PCR and real-time PCR identified more pathogens than blood and tissue cultures; moreover, the sensitivity and specificity of PCR and real-time PCR were more than cultures in most of the studies. The highest sensitivity and specificity were 96% and 100%, respectively. The gram positive bacteria were the most frequent cause of infective endocarditis. The molecular methods enjoy a greater sensitivity compared to the conventional blood culture methods; yet, they are applicable only to the valve tissue of the patients undergoing cardiac valve surgery.

The diagnosis of microorganism involved in infective endocarditis (IE) by polymerase chain reaction (PCR) and real-time PCR: A systematic review.

Science.gov (United States)

Faraji, Reza; Behjati-Ardakani, Mostafa; Moshtaghioun, Seyed Mohammad; Kalantar, Seyed Mehdi; Namayandeh, Seyedeh Mahdieh; Soltani, Mohammadhossien; Emami, Mahmood; Zandi, Hengameh; Firoozabadi, Ali Dehghani; Kazeminasab, Mahmood; Ahmadi, Nastaran; Sarebanhassanabadi, Mohammadtaghi

2018-02-01

Broad-range bacterial rDNA polymerase chain reaction (PCR) followed by sequencing may be identified as the etiology of infective endocarditis (IE) from surgically removed valve tissue; therefore, we reviewed the value of molecular testing in identifying organisms' DNA in the studies conducted until 2016. We searched Google Scholar, Scopus, ScienceDirect, Cochrane, PubMed, and Medline electronic databases without any time limitations up to December 2016 for English studies reporting microorganisms involved in infective endocarditis microbiology using PCR and real-time PCR. Most studies were prospective. Eleven out of 12 studies used valve tissue samples and blood cultures while only 1 study used whole blood. Also, 10 studies used the molecular method of PCR while 2 studies used real-time PCR. Most studies used 16S rDNA gene as the target gene. The bacteria were identified as the most common microorganisms involved in infective endocarditis. Streptococcus spp. and Staphylococcus spp. were, by far, the most predominant bacteria detected. In all studies, PCR and real-time PCR identified more pathogens than blood and tissue cultures; moreover, the sensitivity and specificity of PCR and real-time PCR were more than cultures in most of the studies. The highest sensitivity and specificity were 96% and 100%, respectively. The gram positive bacteria were the most frequent cause of infective endocarditis. The molecular methods enjoy a greater sensitivity compared to the conventional blood culture methods; yet, they are applicable only to the valve tissue of the patients undergoing cardiac valve surgery. Copyright © 2017. Published by Elsevier Taiwan.

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.

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.

Comparative evaluation of the nested ITS PCR against the 18S PCR-RFLP in a survey of bovine trypanosomiasis in Kwale County, Kenya.

Science.gov (United States)

Odongo, Steven; Delespaux, Vincent; Ngotho, Maina; Bekkele, Serkalem Mindaye; Magez, Stefan

2016-09-01

We compared the nested internal transcribed spacer (ITS) PCR and the 18S PCR-RFLP (restriction-fragment length polymorphism) pan-trypanosome assays in a cross-sectional survey of bovine trypanosomiasis in 358 cattle in Kwale County, Kenya. The prevalence of trypanosomiasis as determined by the nested ITS PCR was 19.6% (70/358) and by 18S PCR-RFLP was 16.8% (60/358). Of the pathogenic trypanosomes detected, the prevalence of Trypanosoma congolense and Trypanosoma vivax was greater than that of Trypanosoma simiae The nested ITS PCR detected 83 parasite events, whereas the 18S PCR-RFLP detected 64; however, overall frequencies of infections and the parasite events detected did not differ between the assays (χ(2) = 0.8, df = 1, p > 0.05 and χ(2) = 2.5, df = 1, p > 0.05, respectively). The kappa statistic (0.8) showed good agreement between the tests. The nested ITS PCR and the 18S PCR-RFLP had comparable sensitivity, although the nested ITS PCR was better at detecting mixed infections (χ(2) = 5.4, df = 1, p < 0.05). © 2016 The Author(s).

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.

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.

ANÁLISE DE RISCO DE PRAGUICIDAS EM LEITE CRU E CARACTERIZAÇÃO DO USO EM PROPRIEDADES LEITEIRAS

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Rafael Fagnani

2011-12-01

Full Text Available Este trabalho teve como objetivo a caracterização do uso de praguicidas em propriedades leiteiras no agreste de Pernambuco e estudar a contaminação do leite cru por praguicidas organofosforados (OF e carbamatos (CB, estimando a ingestão diária provável desses resíduos pelo leite. Em 28 propriedades e em dois tanques resfriadores comunitários do agreste pernambucano foram coletadas 30 amostras de leite. A quantificação de praguicidas nas amostras foi realizada por cromatografia gasosa. Doze amostras (40% apresentaram resíduos, sendo seis (20% positivas para OF, cinco (16,7% para CB e uma (3,32% para ambos praguicidas. As médias dos praguicidas detectados no leite em ng/ml foram: 0,04 para coumafós, 0,01 para dimetoato, 0,06 para fention, 0,02 para malation, 0,02 para aldicarb, 0,02 para carbaril e 0,01 para carbofuran. Quando a Ingestão Diária Provável Média dos princípios ativos detectados no leite foi comparada à respectiva Ingestão Diária Aceitável, não houve risco para nenhuma faixa etária, considerando o consumo per capta Brasileiro, pernambucano e o consumo recomendado pelo Ministério da Saúde de leite.

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

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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

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

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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.

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.

Cutaneous and visceral leishmaniasis co-infection in dogs from Rio de Janeiro, Brazil: evaluation by specific PCR and RFLP-PCR assays

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Marize Quinhones Pires

2014-04-01

Full Text Available Introduction During a diagnostic evaluation of canine visceral leishmaniasis (VL, two of seventeen dogs were found to be co-infected by Leishmania (Viannia braziliensis and Leishmania (Leishmania chagasi. Methods Specific polymerase chain reaction (PCR and restriction fragment length polymorphism-PCR (RFLP-PCR assays were performed. Results PCR assays for Leishmania subgenus identification followed by RFLP-PCR analysis in biopsies from cutaneous lesions and the spleen confirmed the presence of Leishmania (Viannia braziliensis and Leishmania (Leishmania chagasi in those fragments. Conclusions This report reinforces the importance of using serological and molecular techniques in the epidemiological surveillance of canine populations in endemic areas in which both diseases are known to co-exist. In such cases, a reassessment of the control measures is required.

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

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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.

Comparação entre diversos antígenos para o diagnóstico de Anaplasma marginale por ELISA Comparison between several antigens for diagnosis of Anaplasma marginale by ELISA

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Carlos A.N. Ramos

2010-01-01

Full Text Available Anaplasmose bovina é uma doença com grande importância nas regiões tropicais e subtropicais do mundo por determinar perdas econômicas devido à mortalidade e redução da produtividade. É causada por Anaplasma marginale, uma riquétsia intraeritrocítica obrigatória cujo controle requer, além de uma vacina eficiente, uma acurada identificação de bovinos cronicamente infectados. Apesar de existirem atualmente diversos métodos de diagnóstico dessa riquétsia, os métodos sorológicos, em particular o ensaio de imunoadsorção enzimática-ELISAs, são os mais utilizados devido à sua versatilidade e praticidade. No entanto, devido ao grande número de antígenos disponíveis, atualmente torna-se necessária uma avaliação para definir quais antígenos apresentam um melhor desempenho no diagnóstico da anaplasmose. Soros de bovinos positivos e negativos para A. marginale por PCR, e soros de animais provenientes do Brasil e Costa Rica, foram testados em ELISAs baseados em MSP1a, MSP2 e MSP5 recombinantes, um pool das três proteínas recombinantes, e antígeno de lisado de corpúsculos iniciais da riquétsia (CI. Utilizando soro de bovinos positivos para A. marginale por PCR, uma maior sensibilidade foi observada no ELISA CI. No entanto, uma maior especificidade, com soro de bovinos negativos a PCR, foi observada com os ELISAs recombinantes. O porcentual de bovinos positivos do Brasil e Costa Rica foi maior com ELISA CI. Razões para essas diferenças são discutidas.Bovine anaplasmosis is a major disease in tropical and subtropical regions of the world by determine economical loss due mortality and productive reduction. The disease is caused by Anaplasma marginale, an intraerythrocytic rickettsia whose control requires, besides an efficient vaccine, the accurate identification of chronically infected cattle. Although the existence of diverse methods of diagnosis of this rickettsia, the serological methods, in particular the enzyme

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)

El plagio, una práctica cotidiana que atenta contra un derecho fundamental: El derecho de autor

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Olga Beatriz Orozco Orozco

2011-01-01

Full Text Available El artículo pretende generar un espacio de reflexión sobre la problemática del plagio desde el punto de vista legal y sus implicaciones en el campo educativo, en especial el ciberplagio, convertido en una verdadera “plaga”, por las facilidades que ofrece la red internet. Por lo tanto, el plagio debe ser detectado y controlado para evitar la violación de un derecho fundamental: el derecho de autor. Desde un análisis de sus causas y consecuencias, se plantean estrategias para prevenir el plagio y de esta manera contribuir al fortalecimiento de la cultura de la legalidad y al fomento de la ética intelectual.

Development of Quantitative Competitive PCR and Absolute Based Real-Time PCR Assays for Quantification of The Butyrate Producing Bacterium: Butyrivibrio fibrisolvens

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Mojtaba Tahmoorespur

2016-04-01

Full Text Available Introduction Butyrivibrio fibrisolvens strains are presently recognized as the major butyrate-producing bacteria found in the rumen and digestive track of many animals and also in the human gut. In this study we reported the development of two DNA based techniques, quantitative competitive (QC PCR and absolute based Real-Time PCR, for enumerating Butyrivibrio fibrisolvens strains. Despite the recent introduction of real-time PCR method for the rapid quantification of the target DNA sequences, use of quantitative competitive PCR (QC-PCR technique continues to play an important role in nucleic acid quantification since it is more cost effective. The procedure relies on the co-amplification of the sequence of interest with a serially diluted synthetic DNA fragment of the known concentration (competitor, using the single set primers. A real-time polymerase chain reaction is a laboratory technique of molecular biology based on the polymerase chain reaction (PCR. It monitors the amplification of a targeted DNA molecule during the PCR. Materials and Methods At first reported species-specific primers targeting the 16S rDNA region of the bacterium Butyrivibrio fibrisolvens were used for amplifying a 213 bp fragment. A DNA competitor differing by 50 bp in length from the 213 bp fragment was constructed and cloned into pTZ57R/T vector. The competitor was quantified by NanoDrop spectrophotometer and serially diluted and co-amplified by PCR with total extracted DNA from rumen fluid samples. PCR products were quantified by photographing agarose gels and analyzed with Image J software and the amount of amplified target DNA was log plotted against the amount of amplified competitor. Coefficient of determination (R2 was used as a criterion of methodology precision. For developing the Real-time PCR technique, the 213 bp fragment was amplified and cloned into pTZ57R/T was used to draw a standard curve. Results and Discussion The specific primers of Butyrivibrio

Ocorrência da infecção por Campylobacter fetus subsp. venerealis e Tritrichomonas foetus em búfalos no estado de Pernambuco, Brasil

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J.M. Borges

Full Text Available RESUMO Objetivou-se com estudo determinar a ocorrência da infecção por Campylobacter fetus subsp. venerealis e Tritrichomonas foetus em búfalos no estado de Pernambuco, Brasil. Foram coletadas 133 amostras biológicas (muco cervicovaginal e raspado prepucial de animais, procedentes de oito propriedades, de diferentes regiões do estado. O material biológico coletado foi transferido para solução salina tamponada (PBS e, posteriormente, inoculado em meios de transporte específicos, Lander para diagnóstico de C. fetus subsp. venerealis e Diamond para T. foetus. Para o diagnóstico das infecções por Campylobacter fetus subsp. venerealis e Tritrichomonas foetus, as amostras foram submetidas à reação em cadeia da polimerase (PCR e cultivadas em meio ágar Columbia acrescido de antibiótico e Diamond, respectivamente. Para pesquisa de C. fetus subsp. venerealis, observou-se uma ocorrência de 1,8% (2/113 de animais positivos no exame microbiológico com confirmação pela PCR. Em relação à procedência, observou-se que 100% das amostras positivas pertenciam a dois machos do mesmo rebanho. Nenhum animal foi positivo na pesquisa de T. foetus. Este é o primeiro registro da infecção por C. fetus subsp. venerealis em búfalos no Brasil. Apesar da baixa ocorrência, recomenda-se adoção de medidas de controle, com o intuito de se evitar a disseminação do agente para outros rebanhos.

Prevalencia y factores de riesgo para infecciones del tracto urinario de inicio en la comunidad causadas por Escherichia coli productor de betalactamasas de espectro extendido en Colombia

Science.gov (United States)

Blanco, Victor M.; Maya, Juan J.; Correa, Adriana; Perenguez, Marcela; Muñoz, Juan S.; Motoa, Gabriel; Pallares, Christian J.; Rosso, Fernando; Matta, Lorena; Celis, Yamile; Garzon, Martha; Villegas, y María V.

2016-01-01

RESUMEN Introducción Las infecciones del tracto urinario (ITU) son frecuentes en la comunidad. Sin embargo, la información de aislamientos resistentes en este contexto es limitada en Latinoamérica. Este estudio tiene como objetivo determinar la prevalencia y los factores de riesgo asociados con ITU de inicio en la comunidad (ITU-IC) causadas por Escherichia coli productor de betalactamasas de espectro extendido (BLEE) en Colombia. Materiales y métodos Entre agosto y diciembre de 2011 se realizó un estudio de casos y controles en 3 instituciones de salud de tercer nivel en Colombia. Se invitó a participar a todos los pacientes admitidos a urgencias con diagnóstico probable de ITU-IC, y se les pidió una muestra de orina. En los aislamien-tos de E. coli se realizaron pruebas confirmatorias para BLEE, susceptibilidad antibiótica, caracterización molecular (PCR en tiempo real para genes bla, repetitive element palindromic PCR [rep-PCR], multilocus sequence typing [MLST] y factores de virulencia por PCR). Se obtuvo información clínica y epidemiológica, y posteriormente se realizó el análisis estadístico. Resultados De los 2.124 pacientes seleccionados, 629 tuvieron un urocultivo positivo, en 431 de estos se aisló E. coli, 54 fueron positivos para BLEE y 29 correspondieron a CTX-M-15. La mayoría de los aislamientos de E. coli productor de BLEE fueron sensibles a ertapenem, fosfomicina y amikacina. La ITU complicada se asoció fuertemente con infecciones por E. coli productor de BLEE (OR = 3,89; IC 95%: 1,10–13,89; p = 0,03). E. coli productor de CTX-M-15 mostró 10 electroferotipos diferentes; de estos, el 65% correspondieron al ST131. La mayoría de estos aislamientos tuvieron 8 de los 9 factores de virulencia analizados. Discusión E. coli portador del gen blaCTX-M-15 asociado al ST131 sigue siendo frecuente en Colombia. La presencia de ITU-IC complicada aumenta el riesgo de tener E. coli productor de BLEE, lo cual debe tenerse en cuenta para ofrecer

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.

Two-temperature LATE-PCR endpoint genotyping

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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

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.

Detection of Histoplasma capsulatum from clinical specimens by cycling probe-based real-time PCR and nested real-time PCR.

Science.gov (United States)

Muraosa, Yasunori; Toyotome, Takahito; Yahiro, Maki; Watanabe, Akira; Shikanai-Yasuda, Maria Aparecida; Kamei, Katsuhiko

2016-05-01

We developed new cycling probe-based real-time PCR and nested real-time PCR assays for the detection of Histoplasma capsulatum that were designed to detect the gene encoding N-acetylated α-linked acidic dipeptidase (NAALADase), which we previously identified as an H. capsulatum antigen reacting with sera from patients with histoplasmosis. Both assays specifically detected the DNAs of all H. capsulatum strains but not those of other fungi or human DNA. The limited of detection (LOD) of the real-time PCR assay was 10 DNA copies when using 10-fold serial dilutions of the standard plasmid DNA and 50 DNA copies when using human serum spiked with standard plasmid DNA. The nested real-time PCR improved the LOD to 5 DNA copies when using human serum spiked with standard plasmid DNA, which represents a 10-fold higher than that observed with the real-time PCR assay. To assess the ability of the two assays to diagnose histoplasmosis, we analyzed a small number of clinical specimens collected from five patients with histoplasmosis, such as sera (n = 4), formalin-fixed paraffin-embedded (FFPE) tissue (n = 4), and bronchoalveolar lavage fluid (BALF) (n = 1). Although clinical sensitivity of the real-time PCR assay was insufficiently sensitive (33%), the nested real-time PCR assay increased the clinical sensitivity (77%), suggesting it has a potential to be a useful method for detecting H. capsulatum DNA in clinical specimens. © 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.

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.

Fusion primer and nested integrated PCR (FPNI-PCR: a new high-efficiency strategy for rapid chromosome walking or flanking sequence cloning

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Wang Zhen

2011-11-01

Full Text Available Abstract Background The advent of genomics-based technologies has revolutionized many fields of biological enquiry. However, chromosome walking or flanking sequence cloning is still a necessary and important procedure to determining gene structure. Such methods are used to identify T-DNA insertion sites and so are especially relevant for organisms where large T-DNA insertion libraries have been created, such as rice and Arabidopsis. The currently available methods for flanking sequence cloning, including the popular TAIL-PCR technique, are relatively laborious and slow. Results Here, we report a simple and effective fusion primer and nested integrated PCR method (FPNI-PCR for the identification and cloning of unknown genomic regions flanked known sequences. In brief, a set of universal primers was designed that consisted of various 15-16 base arbitrary degenerate oligonucleotides. These arbitrary degenerate primers were fused to the 3' end of an adaptor oligonucleotide which provided a known sequence without degenerate nucleotides, thereby forming the fusion primers (FPs. These fusion primers are employed in the first step of an integrated nested PCR strategy which defines the overall FPNI-PCR protocol. In order to demonstrate the efficacy of this novel strategy, we have successfully used it to isolate multiple genomic sequences namely, 21 orthologs of genes in various species of Rosaceace, 4 MYB genes of Rosa rugosa, 3 promoters of transcription factors of Petunia hybrida, and 4 flanking sequences of T-DNA insertion sites in transgenic tobacco lines and 6 specific genes from sequenced genome of rice and Arabidopsis. Conclusions The successful amplification of target products through FPNI-PCR verified that this novel strategy is an effective, low cost and simple procedure. Furthermore, FPNI-PCR represents a more sensitive, rapid and accurate technique than the established TAIL-PCR and hiTAIL-PCR procedures.

Avaliação de dois métodos de extração de DNA de material parafinado para amplificação em PCR Evaluation of two methods of DNA extraction from paraffin-embedded material for PCR amplification

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Luciana Estevam Simonato

2007-04-01

Full Text Available INTRODUÇÃO: Diversos métodos para extração de DNA a partir de tecidos biológicos inclusos em parafina encontram-se descritos na literatura, sendo o sucesso desse procedimento de grande importância para a realização de métodos moleculares de diagnóstico empregando a reação em cadeia da polimerase (PCR. OBJETIVO: Este estudo avaliou dois métodos de extração de DNA de material parafinado, visando à amplificação do DNA genômico pela técnica da PCR. MATERIAL E MÉTODO: Foram utilizadas 35 amostras de casos de carcinoma epidermóide de assoalho bucal diagnosticados e tratados no Centro de Oncologia Bucal da Universidade Estadual Paulista (Unesp. Os métodos de extração de DNA avaliados incluíram: 1. digestão com proteinase K seguida por purificação com Chelex 100® (BioRad; e 2. sistema QIAamp DNA minikit® (Qiagen. O DNA obtido foi quantificado por espectrofotometria e amplificado pela técnica da PCR, utilizando-se oligonucleotídeos iniciadores para betaglobina. RESULTADOS: A concentração de DNA obtido do material extraído com o primeiro método apresentou média de 120,62 ng/µl com razão entre as leituras das absorbâncias 260/280 variando de 0,8 a 1,41. Para as amostras extraídas com o segundo procedimento, o rendimento médio foi de 67,38 ng/µl, no entanto a razão 260/280 variou entre 1,11 e 2,53. O material foi submetido à PCR e, das 35 amostras extraídas com cada método, respectivamente, 29 e 30 apresentaram sinal positivo. CONCLUSÃO: Os dois métodos utilizados para obtenção de DNA de material parafinado apresentaram desempenho semelhante, revelando que ambos têm potencial para auxiliar na prática da biologia molecular diagnóstica, assim como no estudo diagnóstico retrospectivo em material parafinizado.BACKGROUND: There are several methods for DNA extraction from formalin-fixed paraffin-embedded tissues reported in the literature. High success rate on this procedure is important for the use of

Comparison of COBAS AMPLICOR Neissefia gonorrhoeae PCR, including confirmation with N-gonorrhoeae-specific 16S rRNA PCR, with traditional culture

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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

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

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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.

Trypanosoma rangeli: RAPD-PCR and LSSP-PCR analyses of isolates from southeast Brazil and Colombia and their relation with KPI minicircles.

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Marquez, D S; Ramírez, L E; Moreno, J; Pedrosa, A L; Lages-Silva, E

2007-09-01

This study presents the first genetic characterization of five Trypanosoma rangeli isolates from Minas Gerais, in the southeast of Brazil and their comparison with Colombian populations by minicircle classification, RAPD-PCR and LSSP-PCR analyses. Our results demonstrated a homogenous T. rangeli population circulating among Didelphis albiventris as reservoir host in Brazil while heterogeneous populations were found in different regions of Colombia. KP1(+) minicircles were found in 100% isolates from Brazil and in 36.4% of the Colombian samples, whereas the KP2 and KP3 minicircles were detected in both groups. RAPD-PCR and LSSP-PCR profiles revealed a polymorphism within KP1(+) and KP1(-) T. rangeli populations and allowed the division of T. rangeli in two branches. The Brazilian KP1(+) isolates were more homogenous than the KP1(+) isolates from Colombia. The RAPD-PCR were entirely consistent with the distribution of KP1 minicircles while those obtained by LSSP-PCR were associated in 88.9% and 71.4% with KP1(+) and KP1(-) populations, respectively.

Detection of three Allexivirus species infecting garlic in Brazil Detecção de três espécies de Allexivirus que infectam o alho no Brasil

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Péricles de Albuquerque Melo Filho

2004-08-01

Full Text Available Garlic viruses often occur in mixed infections under field conditions. In this study, garlic samples collected in three geographical areas of Brazil were tested by Dot-ELISA for the detection of allexiviruses using monoclonal specific antibodies to detect Garlic virus A (GarV-A, Garlic virus B (GarV-B, Garlic virus C (GarV-C and a polyclonal antiserum able to detect the three virus species mentioned plus Garlic virus D (GarV-D. The detected viruses were biologically isolated by successive passages through Chenopodium quinoa. Reverse Transcriptase Polimerase Chain Reaction (RT-PCR was performed using primers designed from specific regions of the coat protein genes of Japanese allexiviruses available in the Genetic Bank of National Center of Biotechnology Information (NCBI. By these procedures, individual garlic virus genomes were isolated and sequenced. The nucleotide and amino acid sequence analysis and the one with serological data revealed the presence of three distinct allexiviruses GarV-C, GarV-D and a recently described allexivirus, named Garlic mite-borne filamentous virus (GarMbFV, in Brazil.Infecções virais em alho são normalmente causadas por um complexo viral. Neste estudo, um complexo viral de alho, coletado em campo, em três regiões geográficas, foi testado com anti-soros monoclonais específicos para Garlic virus A (GarV-A, Garlic virus B (GarV-B, Garlic virus C (GarV-C e um anti-soro policlonal capaz de detectar os três vírus mencionados e Garlic virus D (GarV-D. Procedeu-se à amplificação por transcriptase reversa-reação em cadeia da polimerase (RT-PCR usando oligonucleotídeos sintetizados a partir de regiões específicas de genes de proteínas capsidiais de allexivirus japoneses e disponíveis no GeneBank (National Center of Biotechnology Information - NCBI. Por esse procedimento, vírus individuais foram isolados e seqüenciados. Os vírus detectados foram biologicamente isolados por meio de sucessivas inocula

Comparison of Direct Sequencing, Real-Time PCR-High Resolution Melt (PCR-HRM) and PCR-Restriction Fragment Length Polymorphism (PCR-RFLP) Analysis for Genotyping of Common Thiopurine Intolerant Variant Alleles NUDT15 c.415C>T and TPMT c.719A>G (TPMT*3C).

Science.gov (United States)

Fong, Wai-Ying; Ho, Chi-Chun; Poon, Wing-Tat

2017-05-12

Thiopurine intolerance and treatment-related toxicity, such as fatal myelosuppression, is related to non-function genetic variants encoding thiopurine S-methyltransferase (TPMT) and Nudix hydrolase 15 (NUDT15). Genetic testing of the common variants NUDT15:NM_018283.2:c.415C>T (Arg139Cys, dbSNP rs116855232 T allele) and TPMT: NM_000367.4:c.719A>G (TPMT*3C, dbSNP rs1142345 G allele) in East Asians including Chinese can potentially prevent treatment-related complications. Two complementary genotyping approaches, real-time PCR-high resolution melt (PCR-HRM) and PCR-restriction fragment length morphism (PCR-RFLP) analysis were evaluated using conventional PCR and Sanger sequencing genotyping as the gold standard. Sixty patient samples were tested, revealing seven patients (11.7%) heterozygous for NUDT15 c.415C>T, one patient homozygous for the variant and one patient heterozygous for the TPMT*3C non-function allele. No patient was found to harbor both variants. In total, nine out of 60 (15%) patients tested had genotypic evidence of thiopurine intolerance, which may require dosage adjustment or alternative medication should they be started on azathioprine, mercaptopurine or thioguanine. The two newly developed assays were more efficient and showed complete concordance (60/60, 100%) compared to the Sanger sequencing results. Accurate and cost-effective genotyping assays by real-time PCR-HRM and PCR-RFLP for NUDT15 c.415C>T and TPMT*3C were successfully developed. Further studies may establish their roles in genotype-informed clinical decision-making in the prevention of morbidity and mortality due to thiopurine intolerance.

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

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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.

Introduction on Using the FastPCR Software and the Related Java Web Tools for PCR and Oligonucleotide Assembly and Analysis.

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Kalendar, Ruslan; Tselykh, Timofey V; Khassenov, Bekbolat; Ramanculov, Erlan M

2017-01-01

This chapter introduces the FastPCR software as an integrated tool environment for PCR primer and probe design, which predicts properties of oligonucleotides based on experimental studies of the PCR efficiency. The software provides comprehensive facilities for designing primers for most PCR applications and their combinations. These include the standard PCR as well as the multiplex, long-distance, inverse, real-time, group-specific, unique, overlap extension PCR for multi-fragments assembling cloning and loop-mediated isothermal amplification (LAMP). It also contains a built-in program to design oligonucleotide sets both for long sequence assembly by ligase chain reaction and for design of amplicons that tile across a region(s) of interest. The software calculates the melting temperature for the standard and degenerate oligonucleotides including locked nucleic acid (LNA) and other modifications. It also provides analyses for a set of primers with the prediction of oligonucleotide properties, dimer and G/C-quadruplex detection, linguistic complexity as well as a primer dilution and resuspension calculator. The program consists of various bioinformatical tools for analysis of sequences with the GC or AT skew, CG% and GA% content, and the purine-pyrimidine skew. It also analyzes the linguistic sequence complexity and performs generation of random DNA sequence as well as restriction endonucleases analysis. The program allows to find or create restriction enzyme recognition sites for coding sequences and supports the clustering of sequences. It performs efficient and complete detection of various repeat types with visual display. The FastPCR software allows the sequence file batch processing that is essential for automation. The program is available for download at http://primerdigital.com/fastpcr.html , and its online version is located at http://primerdigital.com/tools/pcr.html .

TRANSVERSE MODES IN PHASE-CONJUGATION RESONATORS (PCR) WITH FINITE APERTURES (Ⅱ)——FUNDAMENTAL PROPERTIES OF THE TRANSVERSE MODES IN PCR

Institute of Scientific and Technical Information of China (English)

李先枢; 徐家进; 高燕球

1990-01-01

Based on the first part of this paper (Science in China, 33(1990), 982—995), further research has been done on quasi-equivalence relation and asymmetrical character of axisymmetrical phase-conjugatlon resonator(PCR). A series of calculations for axisymmetrical PCR(hundreds of transverse modes in 66 axisymmetrical PCRs) have been carried out, and the results are compared with those of corresponding conventional laser resonators. Fundamental properties of the transverse modes (TEMs) in PCR are summarized. This makes possible a rough estimation of the properties of various TEMs in these simple PCR, including different geometrical structures.

Quantitative analysis of food and feed samples with droplet digital PCR.

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Dany Morisset

Full Text Available In this study, the applicability of droplet digital PCR (ddPCR for routine analysis in food and feed samples was demonstrated with the quantification of genetically modified organisms (GMOs. Real-time quantitative polymerase chain reaction (qPCR is currently used for quantitative molecular analysis of the presence of GMOs in products. However, its use is limited for detecting and quantifying very small numbers of DNA targets, as in some complex food and feed matrices. Using ddPCR duplex assay, we have measured the absolute numbers of MON810 transgene and hmg maize reference gene copies in DNA samples. Key performance parameters of the assay were determined. The ddPCR system is shown to offer precise absolute and relative quantification of targets, without the need for calibration curves. The sensitivity (five target DNA copies of the ddPCR assay compares well with those of individual qPCR assays and of the chamber digital PCR (cdPCR approach. It offers a dynamic range over four orders of magnitude, greater than that of cdPCR. Moreover, when compared to qPCR, the ddPCR assay showed better repeatability at low target concentrations and a greater tolerance to inhibitors. Finally, ddPCR throughput and cost are advantageous relative to those of qPCR for routine GMO quantification. It is thus concluded that ddPCR technology can be applied for routine quantification of GMOs, or any other domain where quantitative analysis of food and feed samples is needed.

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

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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.

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.

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

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

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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.

Absolute quantification of olive oil DNA by droplet digital-PCR (ddPCR): Comparison of isolation and amplification methodologies.

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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.

Avaliação microbiológica e molecular de líquidos articulares e peri-articulares de suínos

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Ana Carolina S. Faria

2011-08-01

Full Text Available No presente estudo coletaram-se 115 amostras de líquido articular e peri-articular de suínos com suspeita clínica de doença articular oriundos de maternidade (30,43%, creche (44,35% e crescimento/terminação (25,22% de Sistemas Intensivos de Produção de Suínos (SIPs para avaliação microbiológica e molecular. Observaram-se 57 (49,5% amostras positivas em pelo menos uma das técnicas. No isolamento microbiano, 39,13% das amostras foram positivas, sendo Streptococcus spp. (19,72%, Arcabobacterium pyogenes (18,13% e Escherichia coli (12,68% os mais frequentes, havendo também a presença de Candida sp. (2,6%. Na técnica de Reação em Cadeia da Polimerase (PCR, em 20% das amostras foram detectados microrganismos com uma maior ocorrência de Mycoplasma hyosinoviae (34,09%, Erysipelotrix tonsilarum (20,45% e Haemophilus parasuis (15,90%. Os microrganismos mais frequentemente isolados em animais com artrite, apresentaram distribuição em todas as faixas etárias, entretanto a fase de crescimento/terminação apresentou maior percentual (69% de amostras positivas. Streptococcus spp. ocorreu em todas as fases sendo o microrganismo mais detectado. M. hyosinoviae foi observado principalmente em animais de creche. Na fase de crescimento/terminação as bactérias predominantes foram A. pyogenes, H. parasuis e E. tonsilarum. Aproximadamente metade dos casos foi negativo o que indica a provável ocorrência de processos degenerativos como a osteocondrose, embora a participação de infecções articulares e peri-articulares possam representar grandes perdas com menor ou maior impacto dependendo da fase de criação. Problemas articulares e/ou peri-articulares de origem infecciosas foram encontrados em todas as propriedades estudadas. O principal agente foi M. hyosynoviae, principalmente na creche, porém não se pode descartar o envolvimento de problemas degenerativos em associação.

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.

Influence of DNA extraction methods, PCR inhibitors and quantification methods on real-time PCR assay of biotechnology-derived traits.

Science.gov (United States)

Demeke, Tigst; Jenkins, G Ronald

2010-03-01

Biotechnology-derived varieties of canola, cotton, corn and soybean are being grown in the USA, Canada and other predominantly grain exporting countries. Although the amount of farmland devoted to production of biotechnology-derived crops continues to increase, lingering concerns that unintended consequences may occur provide the EU and most grain-importing countries with justification to regulate these crops. Legislation in the EU requires traceability of grains/oilseeds, food and feed products, and labelling, when a threshold level of 0.9% w/w of genetically engineered trait is demonstrated to be present in an analytical sample. The GE content is routinely determined by quantitative PCR (qPCR) and plant genomic DNA provides the template for the initial steps in this process. A plethora of DNA extraction methods exist for qPCR applications. Implementing standardized methods for detection of genetically engineered traits is necessary to facilitate grain marketing. The International Organization for Standardization draft standard 21571 identifies detergent-based methods and commercially available kits that are widely used for DNA extraction, but also indicates that adaptations may be necessary depending upon the sample matrix. This review assesses advantages and disadvantages of various commercially available DNA extraction kits, as well as modifications to published cetyltrimethylammonium bromide methods. Inhibitors are a major obstacle for efficient amplification in qPCR. The types of PCR inhibitors and techniques to minimize inhibition are discussed. Finally, accurate quantification of DNA for applications in qPCR is not trivial. Many confounders contribute to differences in analytical measurements when a particular DNA quantification method is applied and different methods do not always provide concordant results on the same DNA sample. How these differences impact measurement uncertainty in qPCR is considered.

Detection of methicillin resistance and slime factor production of Staphylococcus aureus in bovine mastitis Detecção de resistência a meticilina e produção do fator slime por Staphylococcus aureus em mastite bovina

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Alper Ciftci

2009-06-01

Full Text Available This study aimed to detect methicillin resistant and slime producing Staphylococcus aureus in cases of bovine mastitis. A triplex PCR was optimized targetting 16S rRNA, nuc and mecA genes for detection of Staphylococcus species, S. aureus and methicillin resistance, respectively. Furthermore, for detection of slime producing strains, a PCR assay targetting icaA and icaD genes was performed. In this study, 59 strains were detected as S. aureus by both conventional tests and PCR, and 13 of them were found to be methicillin resistant and 4 (30.7% were positive for mecA gene. Although 22 of 59 (37.2% S. aureus isolates were slime-producing in Congo Red Agar, in PCR analysis only 15 were positive for both icaA and icaD genes. Sixteen and 38 out of 59 strains were positive for icaA and icaD gene, respectively. Only 2 of 59 strains were positive for both methicillin resistance and slime producing, phenotypically, suggesting lack of correlation between methicillin resistance and slime production in these isolates. In conclusion, the optimized triplex PCR in this study was useful for rapid and reliable detection of methicillin resistant S. aureus. Furthermore, only PCR targetting icaA and icaD may not sufficient to detect slime production and further studies targetting other ica genes should be conducted for accurate evaluation of slime production characters of S. aureus strains.Este estudo objetivou a detecção de Staphylococcus aureus resistente a meticilina e produtor do fator slime em casos de mastite bovina. Um PCR triplex foi otimizado, com alvo no genes 16SrRNA, nuc e mecA para detecção de Staphylococcus spp, S. aureus e resistencia a meticilina, respectivamente. Para detecção das cepas produtoras do fator slime, empregou-se um PCR com alvo nos genes icaA e icaD. No estudo, 59 cepas foram identificadas como S. aureus por testes convencionais e PCR, sendo 13 resistentes a meticilina e quatro positivas para o gene mecA. Embora 22 das 59 cepas

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...

The PorX response regulator of the Porphyromonas gingivalis PorXY two-component system does not directly regulate the Type IX secretion genes but binds the PorL subunit.

Directory of Open Access Journals (Sweden)

Maxence S Vincent

2016-08-01

Full Text Available The Type IX secretion system (T9SS is a versatile multi-protein complex restricted to bacteria of the Bacteriodetes phylum and responsible for the secretion of surface attachment of diverse proteins that participate to S-layer formation, gliding motility or pathogenesis. The T9SS is poorly characterized but a number of proteins involved in the assembly of the secretion apparatus in the oral pathogen Porphyromonas gingivalis have been identified based on genome substractive analyses. Among these proteins, PorY and PorX encode typical two-component system (TCS sensor and CheY-like response regulator respectively. Although the porX and porY genes do not localize at the same genetic locus, it has been proposed that PorXY form a bona fide TCS. Deletion of the porX in P. gingivalis causes a slight decrease of the expression of a number of other T9SS genes, including sov, porT, porP, porK, porL, porM, porN and porY. Here, we show that PorX and the soluble cytoplasmic domain of PorY interact. Using electrophoretic mobility shift, DNA-protein co-purification and heterologous host expression assays, we showed that PorX does not bind and does not directly regulate expression of the T9SS genes. Finally, we show that PorX interacts with the cytoplasmic domain of PorL, a component of the T9SS membrane core complex and propose that the CheY-like PorX protein might be involved in the dynamics of the T9SS.

Infestation of Palm Trees by Triatomines (Hemiptera: Reduviidae in the State of Bahia, Brazil

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Rodrigo Gurgel-Gonçalves

2012-12-01

Resumo. As palmeiras desempenham papéis importantes como habitats de reprodução e alimentação para triatomíneos silvestres, vetores da doença de Chagas. A ocorrência de triatomíneos em palmeiras peridomiciliares pode aumentar o risco de invasão desses insetos em domicílios e deve ser considerada para desenvolver estratégias de prevenção. Com objetivo de investigar a infestação de palmeiras por triatomíneos no Estado da Bahia e determinar a infecção natural desses insetos por Trypanosoma spp., foram amostradas 183 palmeiras em 12 municípios entre 2006 e 2011 utilizando captura manual e/ou armadilhas adesivas iscadas com camundongos. Os triatomíneos foram detectados em 79 palmeiras (43% das espécies Copernicia prunifera (Mart. Becc., Mauritia flexuosa L. e Attalea spp. (Attalea burretiana Bondar ou Attalea salvadorensis Glassman. Em outras espécies de palmeiras (Syagrus coronata (Mill H.E. Moore, Attalea funifera Mart ex. Spreng e Elaeis guineensis Jacq não foram detectados triatomíneos. Rhodnius neglectus Lent, Triatoma sordida (Stål, e Triatoma pseudomaculata Corrêa & Espínola ocorreram em C. prunifera ao longo do rio São Francisco. No extremo oeste da Bahia, R. neglectus e Psammolestes tertius Lent & Jurberg foram detectados em M. flexuosa, enquanto Triatoma tibiamaculata (Pinto ocorreu em Attalea sp. em áreas urbanas de Salvador. No total, 180 triatomíneos foram capturados, principalmente R. neglectus. A maior taxa de infecção natural (61% foi observada em T. tibiamaculata. Os resultados indicam que pelo menos três espécies de palmeiras são habitats favoráveis para triatomíneos no estado da Bahia e ocorrem no ambiente peridomiciliar, o que pode aumentar a probabilidade de invasão de triatomíneos nas casas.

Rapid and sensitive detection of Feline immunodeficiency virus using an insulated isothermal PCR-based assay with a point-of-need PCR detection platform.

Science.gov (United States)

Wilkes, Rebecca Penrose; Kania, Stephen A; Tsai, Yun-Long; Lee, Pei-Yu Alison; Chang, Hsiu-Hui; Ma, Li-Juan; Chang, Hsiao-Fen Grace; Wang, Hwa-Tang Thomas

2015-07-01

Feline immunodeficiency virus (FIV) is an important infectious agent of cats. Clinical syndromes resulting from FIV infection include immunodeficiency, opportunistic infections, and neoplasia. In our study, a 5' long terminal repeat/gag region-based reverse transcription insulated isothermal polymerase chain reaction (RT-iiPCR) was developed to amplify all known FIV strains to facilitate point-of-need FIV diagnosis. The RT-iiPCR method was applied in a point-of-need PCR detection platform--a field-deployable device capable of generating automatically interpreted RT-iiPCR results from nucleic acids within 1 hr. Limit of detection 95% of FIV RT-iiPCR was calculated to be 95 copies standard in vitro transcription RNA per reaction. Endpoint dilution studies with serial dilutions of an ATCC FIV type strain showed that the sensitivity of lyophilized FIV RT-iiPCR reagent was comparable to that of a reference nested PCR. The established reaction did not amplify any nontargeted feline pathogens, including Felid herpesvirus 1, feline coronavirus, Feline calicivirus, Feline leukemia virus, Mycoplasma haemofelis, and Chlamydophila felis. Based on analysis of 76 clinical samples (including blood and bone marrow) with the FIV RT-iiPCR, test sensitivity was 97.78% (44/45), specificity was 100.00% (31/31), and agreement was 98.65% (75/76), determined against a reference nested-PCR assay. A kappa value of 0.97 indicated excellent correlation between these 2 methods. The lyophilized FIV RT-iiPCR reagent, deployed on a user-friendly portable device, has potential utility for rapid and easy point-of-need detection of FIV in cats. © 2015 The Author(s).

Precisão da medida do limiar anaeróbio por meio do calorímetro portátil Precisión de la medición del umbral anaerobio por medio del calorímetro portátil Measurement precision of the anaerobic threshold by means of a portable calorimeter

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Fernando dos Santos Nogueira

2010-09-01

Full Text Available FUNDAMENTO: Muitos métodos são empregados para determinar o Limiar Anaeróbio (LAn por meio de ergoespirômetros sofisticados. OBJETIVO: Testar a variação no LAn, detectado por modelos matemáticos e de inspeção visual, quando empregado ergoespirômetro de baixo custo e destinado à aplicação clínica. MÉTODOS: Foram voluntários para esse estudo 79 indivíduos aparentemente saudáveis; desses, 57 homens. O VO2máx e o limiar ventilatório foram determinados por calorimetria indireta de circuito aberto. O método eletroenzimático foi empregado para análise da lactacidemia e determinação direta do limiar de lactato (LL. O LAn foi determinado por dois métodos matemáticos (MM SQR e MMslope, baseados nas trocas gasosas, e pelo método de inspeção visual do log-log, para determinação do LL. Dois pesquisadores independentes determinaram o LAn através da inspeção visual de três gráficos, considerando dois métodos (LAn-a= V-slope, EqV; e LAn-b = V-slope, EqV e ExCO2. Os dados foram analisados por meio da estatística paramétrica para determinação das diferenças entre LAn-a versus ExCO2, MM SQR e MMslope; LAn-b versus MM SQR e MMslope; e LL versus LAn-a, LAN-b, MM SQR e MMslope. RESULTADOS: O MMslope foi o único método que apresentou diferença significativa entre o LAn-a e LAn-b (p=0,001, com CV% >15. O LL versus MMslope não apresentou diferença significativa (p=0,274, contudo, observou-se um elevado CV (24%. CONCLUSÃO: Conclui-se que com o equipamento de baixo custo os métodos MM SQR e LAn-a podem ser utilizados para a determinação do LAn. O método MMslope não apresentou precisão satisfatória para ser empregado com esses equipamentos.FUNDAMENTO: Muchos métodos se emplean para que se determine el Umbral Anaerobio (UAn por medio de ergoespirómetros sofisticados. OBJETIVO: Probar la variación en el UAn, detectado por modelos matemáticos y de inspección visual, cuando empleado ergoespirómetro de bajo costo y

Efeitos do treinamento e de uma prova de triathlon em indicadores de lesão muscular e inflamação

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Enrico Fuini Puggina

2016-06-01

Full Text Available Resumo Com o objetivo de verificar a dinâmica de marcadores de lesões musculares e da resposta inflamatória aguda produzidas pelo treinamento e por uma prova de meio ironman em atletas de triathlon durante 12 semanas de treinamento, no presente estudo foram avaliadas as alterações musculares e inflamatórias produzidas pelo treinamento e por um meio-ironman em 12 atletas. Amostras de sangue foram coletadas no início do programa de treinamento (M-1, após 10 semanas de preparação (M-2 e após a competição (M-3. Foram avaliadas as atividades da creatina quinase (CK, lactato desidrogenase (LDH, concentração de IL-6, IL-10, proteína C reativa (PCR e cortisol (C. Foram detectados aumentos em M-3 para a CK (M-1 = 22,25 ± 36,08 UI/L, M-2 = 20,80 ± 42,82 e M-3 = 234,5 ± 135,59 UI/L, LDH (41,71 ± 18,98 U/L, 19,87 ± 16,17 e 191 ± 102,47, PCR (8,42 ± 4,13 mg/L, 5,77 ± 3,54 e 7,62 ± 4,87, IL-6 (77,09 ± 27,86 pg/mL, 93,39 ± 65,2 e 228,48 ± 97,61, IL-10 (88,49 ± 26,36 pg/mL, 89,56 ± 37,99 e 193,31 ± 92,77 e C (14,6 ± 5,92 µg/mL, 23,96 ± 6,44 e 37,47 ± 3,58. A partir desses resultados, conclui-se que os atletas apresentaram aumento dos indicadores de lesões musculares e inflamação apenas após a competição, o que ilustra o efeito agudo dessa prova sobre o organismo desses atletas.

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.

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...

Optimized PCR with sequence specific primers (PCR-SSP for fast and efficient determination of Interleukin-6 Promoter -597/-572/-174Haplotypes

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Bugert Peter

2009-12-01

Full Text Available Abstract Background Interleukin-6 (IL-6 promoter polymorphisms at positions -597(G→A, -572(G→C and -174(G→C were shown to have a clinical impact on different major diseases. At present PCR-SSP protocols for IL-6 -597/-572/-174haplotyping are elaborate and require large amounts of genomic DNA. Findings We describe an improved typing technique requiring a decreased number of PCR-reactions and a reduced PCR-runtime due to optimized PCR-conditions. Conclusion This enables a fast and efficient determination of IL-6 -597/-572/-174haplotypes in clinical diagnosis and further evaluation of IL-6 promoter polymorphisms in larger patient cohorts.

The PorX Response Regulator of the Porphyromonas gingivalis PorXY Two-Component System Does Not Directly Regulate the Type IX Secretion Genes but Binds the PorL Subunit

Science.gov (United States)

Vincent, Maxence S.; Durand, Eric; Cascales, Eric

2016-01-01

The Type IX secretion system (T9SS) is a versatile multi-protein complex restricted to bacteria of the Bacteriodetes phylum and responsible for the secretion or cell surface exposition of diverse proteins that participate to S-layer formation, gliding motility or pathogenesis. The T9SS is poorly characterized but a number of proteins involved in the assembly of the secretion apparatus in the oral pathogen Porphyromonas gingivalis have been identified based on genome substractive analyses. Among these proteins, PorY, and PorX encode typical two-component system (TCS) sensor and CheY-like response regulator respectively. Although the porX and porY genes do not localize at the same genetic locus, it has been proposed that PorXY form a bona fide TCS. Deletion of porX in P. gingivalis causes a slight decrease of the expression of a number of other T9SS genes, including sov, porT, porP, porK, porL, porM, porN, and porY. Here, we show that PorX and the soluble cytoplasmic domain of PorY interact. Using electrophoretic mobility shift, DNA-protein co-purification and heterologous host expression assays, we demonstrate that PorX does not bind T9SS gene promoters and does not directly regulate expression of the T9SS genes. Finally, we show that PorX interacts with the cytoplasmic domain of PorL, a component of the T9SS membrane core complex and propose that the CheY-like PorX protein might be involved in the dynamics of the T9SS. PMID:27630829

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...

Estandarización de la técnica de PCR para amplificar el genoma mitocondrial de las tortugas cabezona (Caretta caretta y carey (Eretmochelys imbricata anidantes del Caribe colombiano

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Javier Hernández Fernández

2013-12-01

Full Text Available Las tortugas marinas, Eretmochelys imbricata y Caretta caretta se encuentran distribuidas en aguas tropicales del Indo-Pacífico y Atlántico y son consideradas especies importantes dentro del ecosistema. Estas tortugas se han catalogado en peligro crítico ya que han sido explotadas por su caparazón, huevos y carne. Las poblaciones de ambas especies se encuentran en un declive poblacional significativo en el Caribe colombiano. Los estudios genéticos del ADN mitocondrial permiten el apoyo de planes de manejo y conservación. En el presente estudio se estandarizaron las mejores condiciones de la técnica de PCR para la amplificación de 22 fragmentos de 800 pb solapadas en 50 pb del mitogenoma de las tortugas E. imbricata y C. caretta. Se diseñaron oligonucleótidos-primers para la amplificación de estos mitogenomas a partir de secuencias previamente descritas de la tortuga verde Chelonia mydas. Se evaluó la concentración de Mg+2, ADN, oligonucleótidos-primers y la temperatura de anillamiento. La reacción estandarizada de PCR se obtuvo en un volumen final de 25 μl, conteniendo 20-70 ng de DNA, 0,5-1 mM de cada oligonucleótidos-primers, 1,5-3,0 mM de Mg+2, 1 U de Taq polimerasa y 0,2 mM de cada dNTP’s. Los parámetros de amplificación optimizados fueron: desnaturalización inicial de 5 min a 94 °C, seguida por 35 ciclos de 94 °C por 1 min, 37-50 °C por 1 min (de acuerdo a los oligonucleótidos-primers y 72 °C por 1 min. Se obtuvo el 63 % de la secuencia de la tortuga carey y el 68 % de la tortuga cabezona. Estas secuencias presentaron un 99-100 % de identidad con las secuencias previamente reportadas.

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...

Diagnóstico de Mycoplasma genitalium por amplificación de los genes MgPa y ARN ribosomal 16S Diagnosis of Mycoplasma genitalium by MgPa and rRNA 16S gene amplification

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Carmen Fernández-Molina

2008-10-01

Full Text Available OBJETIVO: El microorganismo Mycoplasma genitalium se ha relacionado con la uretritis no gonocócica (UNG. La técnica de PCR se ha convertido en el principal método de detección de este patógeno. En consecuencia, debe aplicarse un método de diagnóstico mediante la amplificación de fragmentos de ADN por la técnica PCR. MATERIAL Y MÉTODOS: Se seleccionaron los cebadores MGF-MGR y MgPaF-MgPaR, complementarios de los genes de ARNr 16S y MgPa de M. genitalium, respectivamente. Se efectuaron ensayos de especificidad y sensibilidad y se estudiaron muestras clínicas. RESULTADOS: La PCR con cada grupo de cebadores utilizado fue específica sólo para M. genitalium y la sensibilidad fue mayor con el grupo de cebadores MGF-MGR. En el estudio de 34 muestras clínicas, 18.5% fue positivo a M. genitalium y se encontró un mayor número de muestras positivas al utilizar los cebadores MgPaF-MgPaR. CONCLUSIONES: Debe aplicarse en la práctica clínica el diagnóstico de M. genitalium mediante la amplificación del ADN por PCR en los pacientes con UNG.OBJECTIVE: Mycoplasma genitalium has been associated with nongonococcal urethritis (NGU. Diagnosis by PCR has become the primary detection method for this organism. Thus, diagnosis by DNA amplification using the PCR technique should be utilized. MATERIAL AND METHODS: GMF/GMR and MgpF/MgpR primer pairs, complementary to the M. genitalium 16S rRNA and MgPa genes, respectively, were selected. Specificity and sensibility assays were conducted and clinical samples were studied. RESULTS: The PCR with each primer pair was specific only for M. genitalium, and the sensibility was higher with the GMF/GMR primers. In the study of 34 clinical samples, 18,5% were positive for M. genitalium, with more positive samples when the MgpF/MgpR primers were used. CONCLUSIONS: DNA amplification by PCR should be applied in clinical practice to the diagnosis of M. genitalium in patients with NGU should using.

Quantitative threefold allele-specific PCR (QuanTAS-PCR) for highly sensitive JAK2 V617F mutant allele detection

International Nuclear Information System (INIS)

Zapparoli, Giada V; Jorissen, Robert N; Hewitt, Chelsee A; McBean, Michelle; Westerman, David A; Dobrovic, Alexander

2013-01-01

The JAK2 V617F mutation is the most frequent somatic change in myeloproliferative neoplasms, making it an important tumour-specific marker for diagnostic purposes and for the detection of minimal residual disease. Sensitive quantitative assays are required for both applications, particularly for the monitoring of minimal residual disease, which requires not only high sensitivity but also very high specificity. We developed a highly sensitive probe-free quantitative mutant-allele detection method, Quantitative Threefold Allele-Specific PCR (QuanTAS-PCR), that is performed in a closed-tube system, thus eliminating the manipulation of PCR products. QuantTAS-PCR uses a threefold approach to ensure allele-specific amplification of the mutant sequence: (i) a mutant allele-specific primer, (ii) a 3′dideoxy blocker to suppress false-positive amplification from the wild-type template and (iii) a PCR specificity enhancer, also to suppress false-positive amplification from the wild-type template. Mutant alleles were quantified relative to exon 9 of JAK2. We showed that the addition of the 3′dideoxy blocker suppressed but did not eliminate false-positive amplification from the wild-type template. However, the addition of the PCR specificity enhancer near eliminated false-positive amplification from the wild-type allele. Further discrimination between true and false positives was enabled by using the quantification cycle (Cq) value of a single mutant template as a cut-off point, thus enabling robust distinction between true and false positives. As 10,000 JAK2 templates were used per replicate, the assay had a sensitivity of 1/10 -4 per replicate. Greater sensitivity could be reached by increasing the number of replicates analysed. Variation in replicates when low mutant-allele templates were present necessitated the use of a statistics-based approach to estimate the load of mutant JAK2 copies. QuanTAS-PCR showed comparable quantitative results when validated against a

Comparison of three human papillomavirus DNA detection methods: Next generation sequencing, multiplex-PCR and nested-PCR followed by Sanger based sequencing.

Science.gov (United States)

da Fonseca, Allex Jardim; Galvão, Renata Silva; Miranda, Angelica Espinosa; Ferreira, Luiz Carlos de Lima; Chen, Zigui

2016-05-01

To compare the diagnostic performance for HPV infection using three laboratorial techniques. Ninty-five cervicovaginal samples were randomly selected; each was tested for HPV DNA and genotypes using 3 methods in parallel: Multiplex-PCR, the Nested PCR followed by Sanger sequencing, and the Next_Gen Sequencing (NGS) with two assays (NGS-A1, NGS-A2). The study was approved by the Brazilian National IRB (CONEP protocol 16,800). The prevalence of HPV by the NGS assays was higher than that using the Multiplex-PCR (64.2% vs. 45.2%, respectively; P = 0.001) and the Nested-PCR (64.2% vs. 49.5%, respectively; P = 0.003). NGS also showed better performance in detecting high-risk HPV (HR-HPV) and HPV16. There was a weak interobservers agreement between the results of Multiplex-PCR and Nested-PCR in relation to NGS for the diagnosis of HPV infection, and a moderate correlation for HR-HPV detection. Both NGS assays showed a strong correlation for detection of HPVs (k = 0.86), HR-HPVs (k = 0.91), HPV16 (k = 0.92) and HPV18 (k = 0.91). NGS is more sensitive than the traditional Sanger sequencing and the Multiplex PCR to genotype HPVs, with promising ability to detect multiple infections, and may have the potential to establish an alternative method for the diagnosis and genotyping of HPV. © 2015 Wiley Periodicals, Inc.

Relevancia de la infección natural por Trypanosoma cruzi en triatominos selváticos provenientes de seis municipios de Santander, Colombia

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Sergio Gómez

2016-06-01

genotipificación se realizó mediante PCR del espaciador intergénico del gen mini-exón (SL-IR, que amplifica fragmentos de 350 y 300 pb para T. cruzi I (TcI y T. cruzi II o V-VI (TcII-V-VI, respectivamente. Resultados: Se analizaron 50 especímenes adultos de Panstrongylus geniculatus (64% y Rhodnius pallescens (36% capturados principalmente en el domicilio y peridomicilio, respectivamente. La TI en los triatominos fue de 53,6% al examen directo y de 68,0% mediante PCR de satADN. La TI fue mayor para R. pallescens (77,8% en comparación con P. geniculatus (62,5%. Se observaron altas tasas de infección en triatominos provenientes de Floridablanca (90,9%, Bucaramanga (66,7% y Capitanejo (50,0%. En San Vicente de Chucurí y Lebrija también se detectaron altos índices de infección, no obstante, el número de vectores capturados en estos municipios fue muy bajo (entre 1 y 3. Se determinaron las DTUs de las poblaciones de T. cruzi presentes en el 41,2% de los triatominos selváticos positivos por PCR de satADN. En ambas especies vectoriales se evidenció el predominio de TcI (86% y una alta frecuencia (33% de infección mixta por TcI y TcII-V-VI en R. pallescens. Conclusiones: Se determinó la alta tasa de infección por T. cruzi en triatominos selváticos capturados en los municipios evaluados, lo cual resalta su importancia epidemiológica por la posibilidad de transmisión del parásito por vía oral, aún en zonas de baja endemia o donde no hay reporte de vectores domiciliados. Igualmente, es urgente la implementación de estrategias de control dirigidas a prevenir la intrusión domiciliaria de estos vectores.

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...

Establishing a novel single-copy primer-internal intron-spanning PCR (spiPCR) procedure for the direct detection of gene doping.

Science.gov (United States)

Beiter, Thomas; Zimmermann, Martina; Fragasso, Annunziata; Armeanu, Sorin; Lauer, Ulrich M; Bitzer, Michael; Su, Hua; Young, William L; Niess, Andreas M; Simon, Perikles

2008-01-01

So far, the abuse of gene transfer technology in sport, so-called gene doping, is undetectable. However, recent studies in somatic gene therapy indicate that long-term presence of transgenic DNA (tDNA) following various gene transfer protocols can be found in DNA isolated from whole blood using conventional PCR protocols. Application of these protocols for the direct detection of gene doping would require almost complete knowledge about the sequence of the genetic information that has been transferred. Here, we develop and describe the novel single-copy primer-internal intron-spanning PCR (spiPCR) procedure that overcomes this difficulty. Apart from the interesting perspectives that this spiPCR procedure offers in the fight against gene doping, this technology could also be of interest in biodistribution and biosafety studies for gene therapeutic applications.

Factores relacionados con el control exitoso de un brote por Klebsiella pneumoniae productora de KPC-2 en una unidad de cuidado intensivo en Bogotá, Colombia

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Gisell Bustos-Moya

Full Text Available La resistencia a carbapenémicos en Klebsiella pneumoniae ha aumentado de manera considerable, incrementando las tasas de morbimortalidad. El objetivo de este trabajo fue describir las características epidemiológicas, microbiológicas y las medidas de intervención que permitieron el control exitoso de un brote de Klebsiella pneumoniae productora de KPC-2. Métodos: El estudio se realizó en 2 periodos: el primero durante el brote, con instauración de un protocolo de medidas de intervención; y el segundo, de seguimiento posbrote. Se realizaron pruebas de identificación y susceptibilidad por sistema automatizado, tamización de carbapenemasas por test de Hodge modificado, PCR para detección de los genes bla KPC , bla KPC-2 , NDM-1 y estudio de clonalidad por electroforesis de campos pulsados. Resultados: Durante el brote, se identificaron 18 aislamientos de Klebsiella pneumoniae productora de KPC en 11 pacientes. Tres casos fueron confirmados como infección intrahospitalaria. La técnica de PCR reveló la presencia del gen bla KPC en 21 de 22 aislamientos (pacientes y medio ambiente y se identificó la presencia de un clon con una similitud superior al 75%. En el periodo posbrote los cultivos ambientales y de búsqueda de colonizados fueron negativos. Discusión: Se evidenció un control exitoso del brote producido por un clon. La implementación de un protocolo de intervención y la monitorización de su cumplimiento, la comunicación efectiva y el trabajo en equipo fueron indispensables para evitar su propagación y evitar un comportamiento endémico posbrote.

La influencia de la lateralidad en el rendimiento matemático

OpenAIRE

Vega-Sabaté, Beatriz

2013-01-01

La aparición de dificultades en el rendimiento matemático, es habitual en las aulas . Por ello, el presente trabajo, estudia dicho problema, desde un punto de vista neuropsicológico, concretamente desde la lateralidad. Para ello, se realiza una prueba de lateralidad, en alumnos de 3º de Educación Primaria. Una vez detectado el tipo de lateralidad de cada persona, se relaciona el tipo de lateralidad, con el nivel de rendimiento matemático de cada alumno y su grado de Inteligenci...

Noticias falsas, incorrectas e incompletas: los retos que deben afrontar los periodistas en el proceso de rectificación voluntaria. La experiencia española

OpenAIRE

López Hidalgo, Antonio; Fernández Barrero, María Ángeles

2012-01-01

Los periodistas deben contrastar la información suministrada por sus fuentes informativas. Sin embargo, algunos factores como la precariedad laboral, la premura con la que se elaboran las informaciones y la credibilidad que se le da a las fuentes, principalmente a las institucionales, propician que el periodista no verifique ni contraste sus informaciones. La mayoría de los códigos de conducta recomiendan la obligación de corregir la información tan pronto como se ha detectado el error inv...

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

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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.

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...

Diagnosis of Cutaneous Leishmaniasis by Multiplex PCR

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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.

Effects of erythromycin on γ-glutamyl cysteine synthetase and interleukin-1β in hyperoxia-exposed lung tissue of premature newborn rats

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Cheng Cai

2014-09-01

Full Text Available Objetivo: Explorar o efeito da eritromicina sobre lesões pulmonares induzidas por hiperóxia. Métodos: Uma prole de ratos Sprague-Dawley (SD prematuros com um dia de vida foi dividida aleatoriamente em quatro grupos: grupo 1 ar + cloreto de sódio, grupo 2 ar + eritromicina, grupo 3 hiperóxia + cloreto de sódio e grupo 4 hiperóxia + eritromicina. Com um, sete e 14 dias de exposição, foram detectadas Glutationa (GSH e Interleucina-1 beta (IL-1 beta pelo ensaio imunossorvente ligado à enzima (ELISA, e o ácido bicinconinico (BCA foi utilizado para detectar a proteína GSH. O mRNA da γ-glutamil-cisteina-sintetase (γ-GCS foi detectado por reação em cadeia da polimerase via transcriptase reversa (RT-PCR. Resultados: Comparadas ao grupo 1, as expressões do mRNA da GSH e da γ-GCS no grupo 3 aumentaram significativamente com um e sete dias de exposição (p < 0,05, porém a expressão de mRNA da γ-GCS diminuiu significativamente aos 14 dias; a expressão de IL-1 beta no grupo 3 aumentou significativamente aos 7 dias de exposição (p < 0,05 e diminuiu significativamente aos 14 dias. Comparadas ao grupo 3, as expressões do mRNA da GSH e da γ-GCS no grupo 4 aumentaram significativamente com um, sete e 14 dias de exposição (p < 0,05, porém as expressões de GSH mostraram uma tendência de queda aos 14 dias; a expressão de IL-1 beta no grupo 4 foi reduzida significativamente com um e sete dias de exposição (p < 0,05. Conclusões: As variações de IL-1 beta e GSH mediadas por oxidantes estão envolvidas no desenvolvimento de lesão pulmonar induzida por hiperóxia. A eritromicina poderá regular positivamente a atividade da γ-GCS, aumentando a expressão de GSH, inibindo os níveis de interleucina-1beta mediada por oxidante e aliviando a lesão pulmonar induzida por hiperóxia por meio de um efeito antioxidante.

Role of Helicobacter pylori in stomach cancer after partial gastrectomy for benign ulcer disease Papel del Helicobacter pylori en el cáncer gástrico tras gastrectomía parcial por úlcera benigna

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A. Seoane

2005-11-01

Full Text Available Objective: to determine the prevalence of Helicobacter pylori infection in patients having undergone gastrectomy for non-neoplastic disease who later developed gastric stump cancer. Material and methods: retrospective study of all patients with partial gastrectomy for non-malignant peptic disease who were submitted to an endoscopic exploration between 1995 and 2001. A comparison was made of major clinical and histological characteristics, and the presence of Helicobacter pylori among patients with and without gastric cancer in the stomach remnant. Results: a total of 73 patients were studied in this period. Fifteen patients (20.5% had remnant-stump gastric cancer. All but one were adenocarcinomas (71% intestinal and 29% diffuse, respectively. The average time between diagnosis of gastric cancer and previous gastrectomy was 32 (14-48 years. There was a higher detection rate of Helicobacter pylori in patients with cancer in the gastric remnant (100 vs. 81.5%, respectively, p Objetivo: determinar la prevalencia de la infección por Helicobacter pylori en pacientes gastrectomizados por enfermedad no neoplásica, y que han desarrollado posteriormente cáncer gástrico. Material y métodos: estudio retrospectivo con reclutamiento de todos los pacientes con gastrectomía parcial por enfermedad péptica benigna que han sido sometidos a una exploración endoscópica entre 1995-2001. Se ha realizado una comparación de las principales características clínicas e histológicas y de la presencia de Helicobacter pylori en los pacientes con y sin cáncer del remanente gástrico. Resultados: se han estudiado un total de 73 pacientes en este periodo. Se han encontrado 15 pacientes (20,5% con cáncer en el remanente gástrico, 14 adenocarcinomas (71% tipo intestinal y 29% tipo difuso y un linfoma. El tiempo transcurrido entre el diagnóstico de cáncer gástrico y la gastrectomía previa ha sido de 32 (14-48 años. Se ha detectado un alto porcentaje de infecci

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.

DETECCIÓN Y CUANTIFICACIÓN DE Spongospora subterranea f. sp. subterranea EN PLANTAS SEÑUELO Y CULTIVOS DE PAPA EN COLOMBIA MEDIANTE qPCR

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Nevar Alirio García Bastidas

2013-01-01

Full Text Available La sarna polvosa de la papa (Solanum tuberosum , S. phureja causada por Spongospora subterranea f. sp. subterranea (Sss, es una de las enfermedades más limitantes de este cultivo. En Colombia, se han empleado diferentes métodos de detección asintomática de Sss, incluyendo bioensayos con plantas señuelo, PCR de ITS y pruebas de ELISA. Sin embargo, sus niveles de sensibilidad son bajos o requieren tiempos extensos. Una alternativa para complementar dichas herramientas es la PCR cuantitativa en tiempo real (qPCR. En este trabajo se evaluó dicha técnica utilizando los juegos de cebadores SsTQF1-SsTQR1; Spon421F-Spon494R y SscolF-SscolR (diseñados en este estudio, bajo la metodología de SYBR Green®; mientras que con Taqman® se evaluaron los cebadores SponF-SponR y la sonda SponP. Una vez determinada la funcionalidad de los cebadores, se descartó por inespecificidad, el par Spon421F-Spon494R; para los restantes se realizaron curvas estándar basadas en diluciones seriadas de quistosoros. Las pruebas de qPCR detectaron a Sss en las 20 muestras evaluadas de plantas señuelo de Nicotiana benthamiana y papa, utilizando los cebadores SsTQF1-SsTQR1 (Ct: 10,57- 29,34 y SscolF-SscolR (Ct: 14,39-34,08; mientras que 19 de las muestras fueron positivas con SponF-SponR-SponP (Ct: 15,63-38,93. A partir de 20 muestras de raíces de papa de cultivos de La Unión (Antioquia, Colombia, fue posible detectar el patógeno en 17 de ellas con SscolF-SscolR, estimándose una concentración de 6470 a 1,39 x 1010 quistorosos/mL. Estos resultados indican la ocurrencia de altos niveles de inóculo de Sss en esta región y enfatizan en la necesidad de fortalecer los programas de certificación de tubérculo-semilla en Colombia.

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

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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.

Encefalopatía por priones

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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.

Interlaboratory diagnostic accuracy of a Salmonella specific PCR-based method

DEFF Research Database (Denmark)

Malorny, B.; Hoorfar, Jeffrey; Hugas, M.

2003-01-01

A collaborative study involving four European laboratories was conducted to investigate the diagnostic accuracy of a Salmonella specific PCR-based method, which was evaluated within the European FOOD-PCR project (http://www.pcr.dk). Each laboratory analysed by the PCR a set of independent obtained...... presumably naturally contaminated samples and compared the results with the microbiological culture method. The PCR-based method comprised a preenrichment step in buffered peptone water followed by a thermal cell lysis using a closed tube resin-based method. Artificially contaminated minced beef and whole......-based diagnostic methods and is currently proposed as international standard document....

Detection of HIV and HCV RNA in semen from Brazilian coinfected men using multiplex PCR before and after semen washing Detecção do RNA do HIV e HCV em sêmen de homens brasileiros, usando PCR multiplex antes e depois do "semen washing"

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Cynthia Liliane Motta do Canto

2006-08-01

isolamento de espermatozóides móveis através de método de densidade de gradiente descontínuo e swim-up. Posteriormente, testes para detecção do RNA do HIV e HCV foram aplicados nos sêmens totais e frações seminais obtidas. Em fase pré semen washing, o HIV foi detectado em 100% dos semens totais. Contrariamente, o HCV foi detectado em apenas uma amostra. Em fase pós semen washing, o HIV e HCV não foram detectados em nenhuma das frações seminais. A redução do HIV e do HCV através de semen washing mostra-se um método eficaz a indivíduos co-infectados HIV-HCV, apesar do encontro do HCV no sêmen ser raro.

Comparación de la Respuesta Biológica generada por un sistema de brackets Convencional y brackets de Autoligado.

OpenAIRE

Wilches Buitrago, Liseth; Pontificia Universidad Javeriana; García, Adriana; Pontificia Universidad Javeriana; Quintero, Lina; Pontificia Universidad Javeriana; De Los Reyes, Alfonso; Pontificia Universidad Javeriana; Otero, Liliana; Pontificia Universidad Javeriana

2014-01-01

Antecedentes: No existe evidencia científica suficiente que soporte las ventajas del sistema de fuerzas de autoligado sobre el sistema de fuerzas convencional en ortodoncia. El objetivo de esta investigación  fue Comparar la expresión de OPG y RANKL en el ligamento periodontal de dientes sometido a fuerzas ortodóncicas generadas por un sistema de brackets de autoligado y un sistema convencional. Materiales y Métodos: Se analizó  la expresión de OPG y RANKL mediante RT-PCR en el  ligamento per...

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...

Comparação cromatográfica entre o extrato de Aspidosperma parvifolium e o fitoterápico "Pau-Pereira"

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R.L.R.P. Jácome

Full Text Available A Tintura de "pau-pereira" poderia ser produzida a partir de uma das espécies Aspidosperma parvifolium ou Geissospemun vellosii, espécies conhecidas com o nome popular de "pau-pereira". Análise por CCD e CLAE mostrou que A. parvifolium, uma entre várias Apocynaceae, não é ingrediente da Tintura de "pau-pereira", uma vez que o alcalóide uleína, um marcador químico da espécie, não foi detectado no fitoterápico. Poderia se tratar de G. vellossii, que possui os alcalóides gessospermina, pereirina e velosina, mas o perfil cromatográfico da tintura obtido por CLAE, sugeriu possuir principalmente polifenóis.

Optimization of the Sybr Green real time PCR for the detection of Human Herpes Virus type 6 (HHV-6 Otimização da PCR em tempo real - Sybr Green para detecção do Herpes Vírus Humano tipo 6 (HHV-6

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Cynthia Liliane Motta do Canto

2008-02-01

Full Text Available HHV-6 is the etiological agent of Exanthem subitum which is considered the sixth most frequent disease in infancy. In immuno-compromised hosts, reactivation of latent HHV-6 infection may cause severe acute disease. We developed a Sybr Green Real Time PCR for HHV-6 and compared the results with nested conventional PCR. A 214 pb PCR derived fragment was cloned using pGEM-T easy from Promega system. Subsequently, serial dilutions were made in a pool of negative leucocytes from 10-6 ng/µL (equivalent to 2465.8 molecules/µL to 10-9 (equivalent to 2.46 molecules/µL. Dilutions of the plasmid were amplified by Sybr Green Real Time PCR, using primers HHV3 (5' TTG TGC GGG TCC GTT CCC ATC ATA 3'and HHV4 (5' TCG GGA TAG AAA AAC CTA ATC CCT 3' and by conventional nested PCR using primers HHV1 (outer: 5'CAA TGC TTT TCT AGC CGC CTC TTC 3'; HHV2 (outer: 5' ACA TCT ATA ATT TTA GAC GAT CCC 3'; HHV3 (inner and HHV4 (inner 3'. The detection threshold was determined by plasmid serial dilutions. Threshold for Sybr Green real time PCR was 24.6 molecules/µL and for the nested PCR was 2.46 molecules/µL. We chose the Real Time PCR for diagnosing and quantifying HHV-6 DNA from samples using the new Sybr Green chemistry due to its sensitivity and lower risk of contamination.HHV-6 é o agente etiológico do Exantema Súbito e considerado a sexta doença mais comum na infância. Em indivíduos imunocomprometidos, a reativação da infecção latente pode causar doença aguda ou morte. Padronizamos PCR em Tempo Real utilizando a química Sybr Green na detecção do HHV-6 e comparamos os resultados com a PCR convencional. Um fragmento de 214 pb foi clonado através do kit pGEM-T do sistema Promega. Com este clone, foram feitas diluições seriadas em um pool de leucócitos negativos a partir de 10-6 ng/µL (equivalente a 2465,8 moleculas/µL até 10-9 (equivalente a 2,46 moleculas/µL. As diluições foram amplificadas por PCR em Tempo Real utilizando Sybr Green, com

Control de maquinaria industrial por métodos topográficos

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Julio Roberto Roldán Rodríguez

2016-03-01

Full Text Available La industria requiere en sus programas de mantenimiento industrial del control del funcionamiento de su maquinaria, especialmente aquella que está en operación las 24 horas del día, para poder determinar a tiempo posibles desplazamientos, torsiones, inclinaciones, hundimientos, desalineaciones y desajustes. Para cuantificar estos elementos, los métodos topográficos aplicados de una manera muy especial han demostrado ser muy eficientes. Esto se pudo experimentar en el control que se describe en este artículo, aplicado al sistema de transporte de piezas de cerámica para su cocimiento dentro de un horno de Incesa Standard S.A. El control se realizó con teodolito y cinta métrica de acero para mediciones horizontales y un nivel para las mediciones verticales. Los resultados permitieron a los ingenieros de mantenimiento industrial tomar las medidas correctivas de los defectos detectados en el sistema de transporte.

Human papillomavirus detection and typing using a nested-PCR-RFLP assay.

Science.gov (United States)

Coser, Janaina; Boeira, Thaís da Rocha; Fonseca, André Salvador Kazantzi; Ikuta, Nilo; Lunge, Vagner Ricardo

2011-01-01

It is clinically important to detect and type human papillomavirus (HPV) in a sensitive and specific manner. Development of a nested-polymerase chain reaction-restriction fragment length polymorphism (nested-PCR-RFLP) assay to detect and type HPV based on the analysis of L1 gene. Analysis of published DNA sequence of mucosal HPV types to select sequences of new primers. Design of an original nested-PCR assay using the new primers pair selected and classical MY09/11 primers. HPV detection and typing in cervical samples using the nested-PCR-RFLP assay. The nested-PCR-RFLP assay detected and typed HPV in cervical samples. Of the total of 128 clinical samples submitted to simple PCR and nested-PCR for detection of HPV, 37 (28.9%) were positive for the virus by both methods and 25 samples were positive only by nested-PCR (67.5% increase in detection rate compared with single PCR). All HPV positive samples were effectively typed by RFLP assay. The method of nested-PCR proved to be an effective diagnostic tool for HPV detection and typing.

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

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...

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

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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.

Study comparing human papillomavirus (HPV) real-time multiplex PCR and Hybrid Capture II INNO-LiPA v2 HPV genotyping PCR assays

DEFF Research Database (Denmark)

Iftner, Thomas; Germ, Liesje; Swoyer, Ryan

2009-01-01

methods has not been well characterized. Clinically, cytology is used to establish possible HPV infection. We evaluated the sensitivity and specificity of HPV multiplex PCR assays compared to those of the testing scheme of the Hybrid Capture II (HCII) assay followed by an HPV PCR/line hybridization assay...... (HCII-LiPA v2). SurePath residual samples were split into two aliquots. One aliquot was subjected to HCII testing followed by DNA extraction and LiPA v2 genotyping. The second aliquot was shipped to a second laboratory, where DNA was extracted and HPV multiplex PCR testing was performed. Comparisons...... were evaluated for 15 HPV types common in both assays. A slightly higher proportion of samples tested positive by the HPV multiplex PCR than by the HCII-LiPA v2 assay. The sensitivities of the multiplex PCR assay relative to those of the HCII-LiPA v2 assay for HPV types 6, 11, 16, and 18, for example...

A comparison of QuantStudio™ 3D Digital PCR and ARMS-PCR for measuring plasma EGFR T790M mutations of NSCLC patients

Directory of Open Access Journals (Sweden)

Feng Q

2018-01-01

Full Text Available Qin Feng,1,* Fei Gai,2,* Yaxiong Sang,2 Jie Zhang,3 Ping Wang,1 Yue Wang,1 Bing Liu,2 Dongmei Lin,1 Yang Yu,2 Jian Fang3 1Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education, Department of Pathology, Peking University Cancer Hospital & Institute, 2Oncology Business Division, Beijing Novogene Bioinformatics Technology Co., Ltd, 3Key Laboratory of Carcinogenesis and Translational Research (Ministry of Education, Department of Thoracic Oncology II, Peking University Cancer Hospital & Institute, Beijing, China *These authors contributed equally to this work Background: 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.Purpose: 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.Patients and methods: 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.Results: 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 <0.1%. Limited by the amount of plasma DNA, 17 samples (AF <2.5% and eight samples (T790M- were selected for verification by ARMS-PCR. Four of those samples were verified by NGS as a third verification method. Among the selected 17 positive cases, ten samples presented mutant allele frequency <0.5%, and seven samples presented intermediate mutant allele frequency (0.5%＜AF＜2.5%. However, only three samples (3/17 were identified as positive by ARMS-PCR, namely, P6

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)

Costo efectividad de diferentes estrategias diagnósticas para detección de toxoplasmosis congénita en el recién nacido

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Liliana Chicaíza-Becerra

Full Text Available Objetivo: Estimar la costo-efectividad para Colombia del diagnóstico de toxoplasmosis congénita en el recién nacido de madre con historia de infección durante el embarazo. Métodos. Se construyó un árbol de decisión en TreeAge ® con desenlace casos correctamente identificados. Las alternativas del modelo fueron: i Western Blot (WB; ii IgM e IgA, con confirmación por WB; iii IgG, IgM e IgA con confirmación por WB y seguimiento con IgG. La perspectiva es la del sistema de salud con cifras expresadas en pesos colombianos de 2010 sin descuento. Resultados: La estrategia de menor costo y efectividad fue la i. La estrategia ii es más cara y más efectiva que la i, y la estrategia iii es más cara y efectiva que las anteriores. El costo de detectar un caso adicional correctamente con la estrategia ii en lugar de la i es de $6.189.871. Este resultado es sensible al costo de la prueba de Western Blot y su sensibilidad, así como a la prevalencia de la toxoplasmosis congénita. El costo de detectar un caso adicional correctamente con la estrategia iii en lugar de la ii es de $65.529.979, resultado que es sensible a la prevalencia de la toxoplasmosis congénita, la sensibilidad de la prueba conjunta de IgM e IgA y la sensibilidad del Western Blot. Según el análisis de sensibilidad probabilístico, si la disponibilidad a pagar (DAP por caso detectado adicional es inferior a $6,7 millones de pesos, es más probable que la estrategia costo-efectiva sea la i; quedando entre $6,7 millones de pesos y $74 millones de pesos la ii, y la iii por encima de $74 millones de pesos. Conclusiones: La alternativa costo-efectiva para Colombia dependerá de la disponibilidad a pagar (DAP por caso adicional detectado de toxoplasmosis congénita. La DAP debería tomar en cuenta la efectividad del tratamiento y los costos evitados por las secuelas de la infección.

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.

High-throughput STR analysis for DNA database using direct PCR.

Science.gov (United States)

Sim, Jeong Eun; Park, Su Jeong; Lee, Han Chul; Kim, Se-Yong; Kim, Jong Yeol; Lee, Seung Hwan

2013-07-01

Since the Korean criminal DNA database was launched in 2010, we have focused on establishing an automated DNA database profiling system that analyzes short tandem repeat loci in a high-throughput and cost-effective manner. We established a DNA database profiling system without DNA purification using a direct PCR buffer system. The quality of direct PCR procedures was compared with that of conventional PCR system under their respective optimized conditions. The results revealed not only perfect concordance but also an excellent PCR success rate, good electropherogram quality, and an optimal intra/inter-loci peak height ratio. In particular, the proportion of DNA extraction required due to direct PCR failure could be minimized to <3%. In conclusion, the newly developed direct PCR system can be adopted for automated DNA database profiling systems to replace or supplement conventional PCR system in a time- and cost-saving manner. © 2013 American Academy of Forensic Sciences Published 2013. This article is a U.S. Government work and is in the public domain in the U.S.A.

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.

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…

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.

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.

LA HIPERQUERATOSIS CITOLOGICA COMO PARAMETRO INDIRECTO DE LA INFECCIÓN POR EL VIRUS DEL PAPILOMA HUMANO.

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Victoria Martín Gómez

2009-01-01

Full Text Available Introducción. La hiperqueratosis es una lesión epitelial que histológicamente se caracteriza, por presentar un aumento del grosor de la capa epitelial superficial cornificada, con ausencia de núcleos. Entre las causas que favorecen la aparición de hiperqueratosis, se puede resaltar la reacción del epitelio a los estímulos locales, mecánicos, químicos o infecciosos, siendo más relevante en estos casos el virus del papiloma humano. Objetivo: Conocer la relación que existe entre la presencia de hiperqueratosis en los estudios citológicos de cribado y la posible relación con la infección del virus del Papiloma Humano. Material y métodos: Se han estudiado un total de 2372 extendidos citológicos realizados durante un año en el Servicio de Obstetricia y Ginecología del Hospital Clínico de Salamanca. La edad de las pacientes estaba comprendida entre los 16 y 65 años. La toma citológica realizada mediante la técnica de triple toma se depositaba en medio líquido. Se descartaron las citologías que presentaban alteraciones citológicas, y se valoraron 1125 estudios citológicos considerados como negativos. De estos, 316 presentaron hiperqueratosis. A las pacientes que presentaban hiperqueratosis se les realizó estudio colposcópico y determinación de VPH por PCR. Resultados: En el 27% de las hiperqueratosis, la PCR de VPH fue negativa, el resto, positiva. Se ha podido determinar un VPP del 72% y un VPN del 39%. La sensibilidad fue del 46% y la Especificidad del 81%.

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...

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

Anaplasma marginale infection in cattle from south-western Amazonia Infecção por Anaplasma marginale em bovinos na Amazônia Sul Ocidental, Brasil

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Luciana Gatto Brito

2010-03-01

Full Text Available The present study provides the first epidemiological data regarding infection by Anaplasma marginale in cattle reared in south-western Brazilian Amazonia. One simple procedure was adapted for the extraction of DNA from blood clots collected in seven microregions of Rondônia State and two mesoregions of Acre State. PCR method was used to asses the frequency of A. marginale infections in 4 to12-month-old cattle. The cattle infection was investigated by polymerase chain reaction (PCR using the specific primer "msp5" for A. marginale. The DNA amplifications revealed that the mean frequency of A. marginale infection was 98.6% (1,627/1,650 in samples from Rondonia, and 92.87% (208/225 in samples from Acre. The high frequency of A. marginale infections in 4 to 12-month-old cattle indicate a situation of enzootic stability in the studied areas and are comparable to those detected by immunodiagnosis in different endemic regions in Brazil. The DNA extraction of clotted blood method described here can be used for epidemiological studies on anaplasmosis and other bovine hemoparasites.O presente estudo fornece os primeiros dados epidemiológicos relativos a infecção por Anaplasma marginale em bovinos criados na Amazônia Sul Ocidental brasileira. Foi adaptado um procedimento simples para a extração de DNA a partir de coágulos sanguíneos coletados em sete microrregiões do estado de Rondônia e duas mesoregiões do estado do Acre. A técnica da reação em cadeia da polimerase (PCR foi aplicada para avaliar a freqüência da infecção por A. marginale em bovinos com idade entre 4 e 12 meses. Após a extração do DNA de cada amostra, a infecção nos bovinos foi investigada pela amplificação do gene "msp5" de A. marginale. As técnicas de amplificação do DNA revelaram que a freqüência de infecção por A. marginale foi de 98,6% (1.627/1.650 nas amostras provenientes de Rondônia e de 92,87% (208/225 nas amostras do Acre. A alta freqüência da

MPprimer: a program for reliable multiplex PCR primer design

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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.

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.

A comparative study of digital PCR and real-time qPCR for the detection and quantification of HPV mRNA in sentinel lymph nodes of cervical cancer patients.

Science.gov (United States)

Carow, Katrin; Read, Christina; Häfner, Norman; Runnebaum, Ingo B; Corner, Adam; Dürst, Matthias

2017-10-30

Qualitative analyses showed that the presence of HPV mRNA in sentinel lymph nodes of cervical cancer patients with pN0 status is associated with significantly decreased recurrence free survival. To further address the clinical potential of the strategy and to define prognostic threshold levels it is necessary to use a quantitative assay. Here, we compare two methods of quantification: digital PCR and standard quantitative PCR. Serial dilutions of 5 ng-5 pg RNA (≙ 500-0.5 cells) of the cervical cancer cell line SiHa were prepared in 5 µg RNA of the HPV-negative human keratinocyte cell line HaCaT. Clinical samples consisted of 10 sentinel lymph nodes with varying HPV transcript levels. Reverse transcription of total RNA (5 µg RNA each) was performed in 100 µl and cDNA aliquots were analyzed by qPCR and dPCR. Digital PCR was run in the RainDrop ® Digital PCR system (RainDance Technologies) using a probe-based detection of HPV E6/E7 cDNA PCR products with 11 µl template. qPCR was done using a Rotor Gene Q 5plex HRM (Qiagen) amplifying HPV E6/E7 cDNA in a SYBR Green format with 1 µl template. For the analysis of both, clinical samples and serial dilution samples, dPCR and qPCR showed comparable sensitivity. With regard to reproducibility, both methods differed considerably, especially for low template samples. Here, we found with qPCR a mean variation coefficient of 126% whereas dPCR enabled a significantly lower mean variation coefficient of 40% (p = 0.01). Generally, we saw with dPCR a substantial reduction of subsampling errors, which most likely reflects the large cDNA amounts available for analysis. Compared to real-time PCR, dPCR shows higher reliability. Thus, our HPV mRNA dPCR assay holds promise for the clinical evaluation of occult tumor cells in histologically tumor-free lymph nodes in future studies.

Evaluación de la técnica de MSP-PCR para la caracterización molecular de aislamientos de Rhodotorula mucilaginosa provenientes de la Patagonia noroccidental Assessment of the MSP-PCR technique for the molecular characterization of Rhodotorula mucilaginosa isolates from northwestern Patagonia

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D. Libkind

2007-09-01

Full Text Available La identificación rápida de levaduras de origen ambiental o clínico es de importancia para el estudio de la biodiversidad de estos microorganismos y para la detección de posibles patógenos. Rhodotorula mucilaginosa es una levadura ubicua y pigmentada, capaz de producir infecciones en pacientes inmunocomprometidos. En este trabajo se evaluó la utilidad de la técnica de fingerprinting conocida como MSP-PCR (Micro/Minisatellite-Primed PCR en la caracterización e identificación de aislamientos ambientales de R. mucilaginosa provenientes de la Patagonia noroccidental. Sobre la base de sus caracteres fenotípicos, de un total de 200 levaduras pigmentadas se seleccionaron 110 aislamientos que presuntamente corresponderían a la especie R. mucilaginosa. Se evaluaron los iniciadores (GTG5, (GAC5 y M13 en aislamientos representativos, y se seleccionó el iniciador (GTG5 por ser el que permitió una mejor agrupación de los aislamientos pertenecientes a R. mucilaginosa y una mejor diferenciación de éstos con los de especies filogenéticamente próximas. Utilizando dicho iniciador, el 87% de los aislamientos de R. mucilaginosa presentó un perfil de MSP-PCR similar (> 60% al de la cepa de referencia CBS 316T de R. mucilaginosa. La técnica de MSP-PCR resultó efectiva, tanto para caracterizar e identificar un número elevado de aislamientos ambientales de R. mucilaginosa como para detectar polimorfismos en la especie.The rapid identification of environmental or clinical yeast isolates is important for biodiversity studies and the detection of probable pathogens. Rhodotorula mucilaginosa is a ubiquitous and pigmented yeast capable of infecting immunocompromised patients. In this study, we evaluated the Micro/mini satellite-primed PCR (MSP-PCR fingerprinting method for the characterization and identification of R. mucilaginosa isolates from natural environments in northwestern Patagonia. There were selected 110 putative R. mucilaginosa isolates

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.

Identificação precoce do Transtorno do Espectro Autista por meio da Puericultura em uma Unidade Básica de Saúde

OpenAIRE

Murari, Silvia Cristiane

2014-01-01

O Transtorno do Espectro Autista (TEA) é um transtorno sem etiologia definida. As variáveis que o determinam relacionam-se com a história de vida da criança, principalmente, intercorrências médicas e das interações sociais. O grau de seu comprometimento depende, em parte, do quão precocemente os primeiros sinais de seu desenvolvimento são detectados e de a criança ser encaminhada, o quanto antes, para tratamento adequado. Os primeiros profissionais a terem contado com a criança...

Viability qPCR, a new tool for Legionella risk management.

Science.gov (United States)

Lizana, X; López, A; Benito, S; Agustí, G; Ríos, M; Piqué, N; Marqués, A M; Codony, F

2017-11-01

Viability quantitative Polymerase Chain Reaction (v-qPCR) is a recent analytical approach for only detecting live microorganisms by DNA amplification-based methods This approach is based on the use of a reagent that irreversibly fixes dead cells DNA. In this study, we evaluate the utility of v-qPCR versus culture method for Legionellosis risk management. The present study was performed using 116 real samples. Water samples were simultaneously analysed by culture, v-qPCR and qPCR methods. Results were compared by means of a non-parametric test. In 11.6% of samples using both methods (culture method and v-qPCR) results were positive, in 50.0% of samples both methods gave rise to negative results. As expected, equivalence between methods was not observed in all cases, as in 32.1% of samples positive results were obtained by v-qPCR and all of them gave rise to negative results by culture. Only in 6.3% of samples, with very low Legionella levels, was culture positive and v-qPCR negative. In 3.5% of samples, overgrowth of other bacteria did not allow performing the culture. When comparing both methods, significant differences between culture and v-qPCR were in the samples belonging to the cooling towers-evaporative condensers group. The v-qPCR method detected greater presence and obtained higher concentrations of Legionella spp. (p<0.001). Otherwise, no significant differences between methods were found in the rest of the groups. The v-qPCR method can be used as a quick tool to evaluate Legionellosis risk, especially in cooling towers-evaporative condensers, where this technique can detect higher levels than culture. The combined interpretation of PCR results along with the ratio of live cells is proposed as a tool for understanding the sample context and estimating the Legionellosis risk potential according to 4 levels of hierarchy. Copyright © 2017 Elsevier GmbH. All rights reserved.

Evolución de la prevalencia de infección por virus de la inmunodeficiencia humana en población reclusa al ingreso en prisión durante el período 1991-1995

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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

El artículo científico para revista académica: Pautas para su planificación y edición de acuerdo con el modelo APA

OpenAIRE

Camacho Villalobos, María Elena; Rojas Porras, Marta Eugenia; Rojas Blanco, Lillyam

2014-01-01

A partir de la experiencia de las autoras como directoras y editoras de revistas académicas, en comités editoriales, en arbitrajes de textos, en cursos impartidos para la redacción de artículos y sobre la escritura en general, y en la revisión filológica, se propone este artículo. En estas experie ncias han detectado diversas dificultades en la escritura de artículos, por lo cual plantean los indicadores de calidad e información pertinente para la escogencia d...

Variabilidad en la lectura de radiografía de tórax en el diagnóstico de neumoconiosis del minero de carbón

OpenAIRE

Gómez Ávila, Germán Darío

2014-01-01

Diversas clasificaciones radiológicas han sido desarrolladas para apoyar el diagnóstico de la neumoconiosis. El propósito de este estudio fue comparar la concordancia de los hallazgos radiográficos relacionados con el diagnóstico de neumoconiosis detectados en la lectura de la radiografía de tórax realizada por medico entrenado en sistema de clasificación de la Organización Internacional del Trabajo (OIT 2.000) y radiólogo especialista. Metodología: Estudio descriptivo. Se comparó la lectura ...

164. Extracción endovascular de dispositivos cardíacos infectados

OpenAIRE

Gutiérrez Carretero, E.; Hernández Fernández, A.; Borrego Domínguez, J.M.; Alarcón, A.; Eslava, J.; Bibiloni Lage, I.; Ruiz Solano, E.; Romero Rodríguez, N.

2010-01-01

La tasa de infección publicada en la literatura para implantes de marcapasos y desfibriladores es del 1-7%, siendo en nuestro hospital de 1,5% para marcapasos y 3% para desfibrilador automático implantable (DAI), con una mortalidad del 10%. En un estudio realizado por la Sociedad Andaluza de Enfermedades Infecciosas sobre seis hospitales andaluces, se han detectado 243 infecciones de MP/DAI entre 1998-2008, de las cuales un 44,5% han sido infecciones locales y en un 55,5% infecciones sistémic...

Evaluación molecular y fenotípica de la sensibilidad al fungicida fenamidone en aislamientos de Peronospora sparsa

OpenAIRE

Argel Luz Edith; Marín Mauricio; Cotes José Miguel; Jaramillo Sonia; Guzmán Edgar

2007-01-01

El Mildeo velloso es uno de los mayores limitantes del cultivo de la rosa en Colombia. Esta enfermedad es causada por el Oomycete holobiótrofo Peronospora sparsa que fue detectado en la década de 1970 en la Sabana de Bogotá. En los últimos años su efecto negativo sobre la floricultura aumentó conduciendo a los cultivadores al empleo excesivo de fungicidas con acción sistémica, muchos de los cuales no cuentan con estudios de líneas base de sensibilid...

Poluentes Orgânicos Emergentes: Impactos e Soluções para a Saúde Humana e o Meio Ambiente

OpenAIRE

Nascimento, Lucas X; Trindade Araújo, Reginaldo; Giraldez Alvarez, Lisandro D

2015-01-01

Os Poluentes Orgânicos Emergentes (POE) formam um grupo muito heterogêneo de substâncias cuja característica comum é que causam efeitos adversos sobre os organismos aquáticos, e por isso devem ser removidos do ambiente. Esta revisão fornece uma visão geral dos contaminantes emergentes detectados no ambiente aquático, e analisa os trabalhos mais atuais, a fim de refletir sobre as diversas fontes dos contaminantes, os métodos de tratamento e os riscos para a saúde humana e para o meio ambiente,...

Papel de la enfermería en la valoración y estudios oftalmológicos en pediatría en atención primaria

OpenAIRE

Pérez Andrés, Irene

2013-01-01

La oftalmología pediátrica tiene gran importancia ya que cualquier problema ocular si es detectado precozmente puede curarse y ó evitar su progresión en esta edad. Por ello, resulta fundamental realizar una valoración y estudios oftalmológicos de forma adecuada en atenció primaria, ya que suele ser el primer lugar donde se detectan patologías oculares.También es necesario conocer las nfermedades oftalmológicas más frecuentes y saber actuar correctamente en las urgencias oftalmológicas. Enf...

Gangrena de Fournier: infección necrotizante de los genitales externos y del perine, a propósito de 5 casos

OpenAIRE

Haydee Wong Arocha; Alberto Elejalde Hernández; Armando Fleites Castro

1995-01-01

La gangrena de Fournier es una forma de gangrena sinergística y frecuente del revestimiento cutáneo de los órganos genitales externos y del periné. Esta afección descrita por Fournier en 1884 recibe varias denominaciones como: gangrena escrotal y fulminante. La incidencia de esta entidad es poco frecuente, no obstante, dentro de las urgencias urológicas de nuestro Servicio de Urología, hemos apreciado que en los últimos 6 meses se han detectado un total de 5 casos para el 18,5 %. En los casos...

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.

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

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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

Applicazioni della PCR e PCR in situ nella diagnosi di infezioni batteriche e virali da biopsie fissate in formalina e incluse in paraffina

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Stefania Cazzavillan

2003-03-01

Full Text Available In situ PCR, amplification of target DNA sequences in fixed cells, is a very useful molecular biology tecnique with potential to combine the high sensitivity of tube PCR with the precise anatomical localization of the targeted bsequences. It allows the study of low copy viral or bacterial DNA. In this study we document the utility of directin situ PCR with single primer pair by applying it to 3 infectious agents in different model systems: Borrelia burgdorferi in 5 Eritema migrans lesions and 55 primitive cutaneous B cell lymphomas, Chlamydia pneumoniae in 200 autoptic atheromasic lesions, and Papilloma virus in 20 CIN 1 (mild cervical dysplasia. In situ PCR seems to be a very promising tecnique; however, the prerequisite for the success of in situ PCR is conditioned by optimal standardization of the key variables which, on the other hand, are influenced by tissue composition.

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.

Real-Time PCR using a PCR Microchip with Integrated Thermal System and Polymer Waveguides for the Detection of Campylobacter jejuni

DEFF Research Database (Denmark)

Wang, Zhenyu; Sekulovic, Andrea; Kutter, Jörg Peter

2006-01-01

A novel real-time PCR microchip platform with integrated thermal system and polymer waveguides has been developed. By using the integrated optical system of the real-time PCR chip, cadF – a virulence gene of Campylobacter jejuni, could specifically be detected. Two different DNA binding dyes, SYTOX...

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

Diagnosis of the fragile X syndrome in males using methylation-specific PCR of the FMRI locus

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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

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....

Development and validation of a quantitative PCR assay for Ichthyophonus spp.

Science.gov (United States)

White, Vanessa C; Morado, J Frank; Crosson, Lisa M; Vadopalas, Brent; Friedman, Carolyn S

2013-04-29

Members of the genus Ichthyophonus are trophically transmitted, cosmopolitan parasites that affect numerous fish species worldwide. A quantitative PCR (qPCR) assay specific for genus Ichthyophonus 18S ribosomal DNA was developed for parasite detection and surveillance. The new assay was tested for precision, repeatability, reproducibility, and both analytical sensitivity and specificity. Diagnostic sensitivity and specificity were estimated using tissue samples from a wild population of walleye pollock Theragra chalcogramma. Ichthyophonus sp. presence in tissue samples was determined by qPCR, conventional PCR (cPCR), and histology. Parasite prevalence estimates varied depending upon the detection method employed and tissue type tested. qPCR identified the greatest number of Ichthyophonus sp.-positive cases when applied to walleye pollock skeletal muscle. The qPCR assay proved sensitive and specific for Ichthyophonus spp. DNA, but like cPCR, is only a proxy for infection. When compared to cPCR, qPCR possesses added benefits of parasite DNA quantification and a 100-fold increase in analytical sensitivity. Because this novel assay is specific for known members of the genus, it is likely appropriate for detecting Ichthyophonus spp. DNA in various hosts from multiple regions. However, species-level identification and isotype variability would require DNA sequencing. In addition to distribution and prevalence applications, this assay could be modified and adapted for use with zooplankton or environmental samples. Such applications could aid in investigating alternate routes of transmission and life history strategies typical to members of the genus Ichthyophonus.

Technical aspects and recommendations for single-cell qPCR.

Science.gov (United States)

Ståhlberg, Anders; Kubista, Mikael

2018-02-01

Single cells are basic physiological and biological units that can function individually as well as in groups in tissues and organs. It is central to identify, characterize and profile single cells at molecular level to be able to distinguish different kinds, to understand their functions and determine how they interact with each other. During the last decade several technologies for single-cell profiling have been developed and used in various applications, revealing many novel findings. Quantitative PCR (qPCR) is one of the most developed methods for single-cell profiling that can be used to interrogate several analytes, including DNA, RNA and protein. Single-cell qPCR has the potential to become routine methodology but the technique is still challenging, as it involves several experimental steps and few molecules are handled. Here, we discuss technical aspects and provide recommendation for single-cell qPCR analysis. The workflow includes experimental design, sample preparation, single-cell collection, direct lysis, reverse transcription, preamplification, qPCR and data analysis. Detailed reporting and sharing of experimental details and data will promote further development and make validation studies possible. Efforts aiming to standardize single-cell qPCR open up means to move single-cell analysis from specialized research settings to standard research laboratories. Copyright © 2017 Elsevier Ltd. All rights reserved.

Interferência da fração mineral na estimativa do grau de humificação da matéria orgânica em agregados organominerais por ressonância paramagnética eletrônica

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C. Bayer

2000-03-01

Full Text Available A concentração de radicais livres semiquinona (CRLS, determinada por ressonância paramagnética eletrônica (EPR, é considerada um índice do grau de humificação, sendo uma importante determinação em estudos qualitativos da matéria orgânica do solo. Neste trabalho, avaliou-se a interferência da fração mineral na quantificação da CRLS em agregados organominerais 20-53, 2-20 e < 2 ∝m de Podzólico Vermelho-Amarelo, Podzólico Vermelho-Escuro e Latossolo Roxo. A CRLS foi determinada pela área do sinal, estimada pela aproximação intensidade do sinal (I, em cm, multiplicada pela sua largura de linha ao quadrado (∆H², em Gauss. Os parâmetros espectrais I e ∆H foram obtidos em espectros de EPR com e sem interferência da fração mineral. No Podzólico Vermelho-Amarelo e no Podzólico Vermelho-Escuro, foram detectados dois sinais de radicais livres, um com um valor g 2,004 e largura de linha de 5-6 G, típico de radicais livres semiquinona, outro com um valor g 2,000 e largura de linha de 2-3 G, associado à fração mineral, especificamente ao quartzo (SiO2, como confirmado posteriormente por análise de amostra purificada. Nestes solos, a interferência da fração mineral na obtenção dos parâmetros I e ∆H resultou num erro na estimativa da CRLS de -7 a +488%, comparativamente às quantificações realizadas a partir dos espectros sem interferência da fração mineral. No Latossolo Roxo, os altos teores de Fe3+ não permitiram detectar os sinais dos radicais livres semiquinona por causa da sobreposição dos sinais do metal. A eliminação da interferência da fração mineral demonstrou ser um pré-requisito fundamental no estudo da matéria orgânica por EPR em agregados organominerais, para a qual são sugeridos alguns procedimentos alternativos.

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.

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.

Utilidad de la reacción en cadena de la polimerasa en el diagnóstico de infección congénita por citomegalovirus: a propósito de un caso de meningitis aséptica

Directory of Open Access Journals (Sweden)

Yolanda Cifuentes-Cifuentes

Full Text Available Se informa del caso de un recién nacido que presentó trombocitopenia, hematuria y proteinuria. En el líquido cefalorraquídeo tenía aumento de proteínas y leucocitos, VDRL no reactiva. La madre tenía historia de sífilis gestacional. Las determinaciones de IgM para citomegalovirus, rubéola, Toxoplasma , herpes i y ii fueron negativas por lo que se consideró caso de sífilis congénita con compromiso de sistema nervioso central. Por persistir la trombocitopenia después del tratamiento, se tomó muestra de sangre para PCR para citomegalovirus, encontrándose 181.171 copias/ml. Se dio tratamiento con ganciclovir intravenoso 12 mg/kg de peso durante 21 días y solución al 10% de inmunoglobulina humana hiperinmune para citomegalovirus administrada así: 4 ml/kg de peso los días 0, 4 y 8, seguido de 2 ml/kg de peso los días 12 y 16. La evolución fue satisfactoria. Se evidenció la utilidad de PCR en el diagnóstico de infección congénita por citomegalovirus.

Shape based kinetic outlier detection in real-time PCR

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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.

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

Incidence of pulmonary aspergillosis and correlation of conventional diagnostic methods with nested PCR and real-time PCR assay using BAL fluid in intensive care unit patients.

Science.gov (United States)

Zarrinfar, Hossein; Makimura, Koichi; Satoh, Kazuo; Khodadadi, Hossein; Mirhendi, Hossein

2013-05-01

Although the incidence of invasive aspergillosis in the intensive care unit (ICU) is scarce, it has emerged as major problems in critically ill patients. In this study, the incidence of pulmonary aspergillosis (PA) in ICU patients has evaluated and direct microscopy and culture has compared with nested polymerase chain reaction (PCR) and real-time PCR for detection of Aspergillus fumigatus and A. flavus in bronchoalveolar lavage (BAL) samples of the patients. Thirty BAL samples obtained from ICU patients during a 16-month period were subjected to direct examinations on 20% potassium hydroxide (KOH) and culture on two culture media. Nested PCR targeting internal transcribed spacer ribosomal DNA and TaqMan real-time PCR assay targeting β-tubulin gene were used for the detection of A. fumigatus and A. flavus. Of 30 patients, 60% were men and 40% were women. The diagnosis of invasive PA was probable in 1 (3%), possible in 11 (37%), and not IPA in 18 (60%). Nine samples were positive in nested PCR including seven samples by A. flavus and two by A. fumigatus specific primers. The lowest amount of DNA that TaqMan real-time PCR could detect was ≥40 copy numbers. Only one of the samples had a positive result of A. flavus real-time PCR with Ct value of 37.5. Although a significant number of specimens were positive in nested PCR, results of this study showed that establishment of a correlation between the conventional methods with nested PCR and real-time PCR needs more data confirmed by a prospective study with a larger sample group. © 2013 Wiley Periodicals, Inc.

Monitoramento da presença de Mycoplasma hyopneumoniae em granjas de suínos durante a implementação de programas de erradicação Monitoring the presence of Mycoplasma hyopneumoniae in swine farms during the implementation of eradication programs

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Pablo Jesús Tamiozzo

2011-04-01

Full Text Available O objetivo deste estudo foi monitorar a presença de M. hyopneumoniae em granjas suínas durante a implementação de programas de erradicação utilizando diferentes técnicas de diagnóstico focalizando no PCR. Trabalhou-se com uma empresa que possuía três granjas, uma parto-terminação (390 matrizes, uma múltiplo-sítio (4100 matrizes e uma nova granja que povoava suas novas instalações. Nas duas primeiras, foi desenvolvido um programa de despovoamento parcial para erradicar a pneumonia enzoótica suína, a última foi povoada pelos suínos dos anteriores após a erradicação. Nos três rebanhos, os suínos foram monitorados por: sorologia (ELISA, PCR, lesões pulmonares macro e microscópicas e a presença de tosse não produtiva. A ausência de tosse, a baixa porcentagem de suínos soropositivos na fase de terminação e a baixa proporção de lesões pulmonares no abate sugerem que a pneumonia enzoótica suína foi erradicada, mas não o agente causativo -M. hyopneumoniae- cujo DNA foi detectado pela PCR, mostrando diferentes comportamentos de acordo com o rebanho.The aim of this study was to monitor the presence of M. hyopneumoniae in pig farms during the implementation of eradication programs using different diagnostic techniques focusing on PCR. They worked with a company owner of three farms, a farrow-to-finish (390 sows, a multiple-site (4100 sows and a new one that was populated its new facility. In the first two were developed a partial depopulation program to eradicate swine enzootic pneumonia, the latter one was populated with pigs after the previous eradication. In the three farms, the pigs were monitored by: serology (ELISA, PCR, macroscopic and microscopic lung lesions and the presence of non-productive cough. The absence of cough, low percentage of seropositive pigs in the finishing stage and the low proportion of lung lesions at slaughter suggest that swine enzootic pneumonia was eradicated, but not the causative agent

Interlaboratory study of DNA extraction from multiple ground samples, multiplex real-time PCR, and multiplex qualitative PCR for individual kernel detection system of genetically modified maize.

Science.gov (United States)

Akiyama, Hiroshi; Sakata, Kozue; Makiyma, Daiki; Nakamura, Kosuke; Teshima, Reiko; Nakashima, Akie; Ogawa, Asako; Yamagishi, Toru; Futo, Satoshi; Oguchi, Taichi; Mano, Junichi; Kitta, Kazumi

2011-01-01

In many countries, the labeling of grains, feed, and foodstuff is mandatory if the genetically modified (GM) organism content exceeds a certain level of approved GM varieties. We previously developed an individual kernel detection system consisting of grinding individual kernels, DNA extraction from the individually ground kernels, GM detection using multiplex real-time PCR, and GM event detection using multiplex qualitative PCR to analyze the precise commingling level and varieties of GM maize in real sample grains. We performed the interlaboratory study of the DNA extraction with multiple ground samples, multiplex real-time PCR detection, and multiplex qualitative PCR detection to evaluate its applicability, practicality, and ruggedness for the individual kernel detection system of GM maize. DNA extraction with multiple ground samples, multiplex real-time PCR, and multiplex qualitative PCR were evaluated by five laboratories in Japan, and all results from these laboratories were consistent with the expected results in terms of the commingling level and event analysis. Thus, the DNA extraction with multiple ground samples, multiplex real-time PCR, and multiplex qualitative PCR for the individual kernel detection system is applicable and practicable in a laboratory to regulate the commingling level of GM maize grain for GM samples, including stacked GM maize.

Modified DNA extraction for rapid PCR detection of methicillin-resistant staphylococci

International Nuclear Information System (INIS)

Japoni, A.; Alborzi, A.; Rasouli, M.; Pourabbas, B.

2004-01-01

Nosocomial infection caused by methicillin-resistant staphylococci poses a serious problem in many countries. The aim of this study was to rapidly and reliably detect methicillin-resistant-staphylococci in order to suggest appropriate therapy. The presence or absence of the methicillin-resistance gene in 115 clinical isolates of staphylococcus aureus and 50 isolates of coagulase negative staphylococci was examined by normal PCR. DNA extraction for PCR performance was then modified by omission of achromopeptadiase and proteinase K digestion, phenol/chloroform extraction and ethanol precipitation. All isolates with Mic>8 μ g/ml showed positive PCR. No differences in PCR detection have been observed when normal and modified DNA extractions have been performed. Our modified DNA extraction can quickly detect methicillin-resistant staphylococci by PCR. The advantage of rapid DNA extraction extends to both reduction of time and cost of PCR performance. This modified DNA extraction is suitable for different PCR detection, when staphylococci are the subject of DNA analysis

Estimación de la evapotranspiración de dos cultivares de Gerbera jamesonii en condiciones de hidroponia.

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Libertad Mascarini

2003-06-01

Full Text Available La producción comercial de Gerbera jamesonii para flor de corte ocupa un lugar destacado a nivel mundial. En Argentina la superficie de cultivo bajo invernadero está en aumento y no cubre la demanda del mercado. La introducción de nuevas tecnologías de riego y cultivo que están llevando a cabo los productores, requiere del conocimiento de las necesidades hídricas del cultivo para que el sistema resulte eficiente. El objetivo de este trabajo es caracterizar el consumo hídrico de dos cultivares de Gerbera en hidroponía, mediante el ajuste de modelos de uso corriente, para el área de influencia de la ciudad de Buenos Aires, en el ciclo de primavera. En este ensayo se evalúa el consumo de agua mediante bandejas de drenaje a fin dedeterminar el coeficiente del cultivo (Kc para cada una de las fórmulas empleadas; también secaracteriza el clima del invernadero a fin de hallar los valores de evapotranspiración potencial o de referencia (Eto para esa situación empleando fórmulas de uso corriente y se selecciona la relación entre Eto y Kc que determine la evapotranspiración del cultivo (Etc que mejor se ajuste al consumo medido. No se han detectado diferencias significativas entre ETc y ETo calculada por el método de Penman-Monteith, modificado por FAO, siendo el que mejor se ajusta a la estimación de Eto en las condiciones de invernadero; por el contrario, se han hallado diferencias significativas utilizando el método de Blaney-Criddle. Asimismo, se han detectado diferencias significativas entre la ETc de las dos variedades ensayadas.

An optimized one-tube, semi-nested PCR assay for Paracoccidioides brasiliensis detection.

Science.gov (United States)

Pitz, Amanda de Faveri; Koishi, Andrea Cristine; Tavares, Eliandro Reis; Andrade, Fábio Goulart de; Loth, Eduardo Alexandre; Gandra, Rinaldo Ferreira; Venancio, Emerson José

2013-01-01

Herein, we report a one-tube, semi-nested-polymerase chain reaction (OTsn-PCR) assay for the detection of Paracoccidioides brasiliensis. We developed the OTsn-PCR assay for the detection of P. brasiliensis in clinical specimens and compared it with other PCR methods. The OTsn-PCR assay was positive for all clinical samples, and the detection limit was better or equivalent to the other nested or semi-nested PCR methods for P. brasiliensis detection. The OTsn-PCR assay described in this paper has a detection limit similar to other reactions for the molecular detection of P. brasiliensis, but this approach is faster and less prone to contamination than other conventional nested or semi-nested PCR assays.

Polimorfismo genético de beta-lactoglobulina y alphalactoalbúmina en el ganado criollo colombiano, mediante PCR-SSCP

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Jaime A Rosero-Alpala

2011-12-01

Full Text Available La población de ganado criollo colombiano ha venido presentando una inquietante disminución al pasar de 23.415 ejemplares en 1999 a 20.102 en 2003. A pesar de los esfuerzos por recuperar las razas criollas el panorama para su conservación es incierto, por tanto la búsqueda de caracteres deseables puede contribuir a su valoración y conservación. Los genes relacionados con el mejoramiento de la calidad de la leche producida por estas razas se consideran de gran importancia en la industria láctea, por tal razón y con el objetivo de caracterizar los genes beta-lactoglobulina y alpha-lactoalbúmina se analizaron 30 muestras de sangre de cada una de las razas criollas (Blanco Orejinegro, Caqueteño, Casanareño, Costeño con cuernos, Chino Santandereano, Hartón del Valle, Romosinuano y Sanmartinero, dos razas sintéticas colombianas (Lucerna y Velásquez y dos razas foráneas (Holstein y Brahman. Se amplificaron fragmentos de 262pb para beta-lactoglobulina (b-LG y de 166 pb para alpha-lactoalbúmina (a-LA que se genotipificaron mediante PCR-SSCP. El promedio de la frecuencia para b-LG A y b-LG B fue de 0.46 ± 0.020 y de 0.53 ± 0.020, respectivamente, y de 0.35 ± 0.019 para a-LA A y 0.64 ± 0.019 para a-LA B. El promedio de diversidad genética (He para b-LG fue 0.498 y de 0.455 para a-LA. Los ganados criollos representan una base genética valiosa, como alternativa para mejorar genéticamente los hatos destinados a la producción de leche con mejores características en calidad para la industria láctea.

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 ...