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1
artículo
Publicado 2011
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Se estandarizó y validó la técnica de Transcriptasa Reversa - Reacción en Cadena de la Polimerasa (RT-T-PCR) en tiempo real para el diagnóstico de la Peste Porcina Clásica (PPC). Se utilizó extractos de tonsilas y nódulos linfáticos de porcinos positivos (n=36) y negativos (n=30) a PPC mediante inmunofluorescencia (IF). La extracción de ARN viral, síntesis del ADN complementario y PCR en tiempo real se realizaron utilizando kits comerciales. Se utilizaron tres pares de cebadores para amplificar una región conservada (5’UTR) del virus PPC (VPPC) y del panpestivirus (virus de la DVB y EF) y una región codificante de la proteína E2 del VPPC frente a cepas de referencia del VPPC Alfort/187, Brescia y cepa China vacunal como controles positivos y cepas de los virus diarrea viral bovina, enfermedad de la frontera y rotavirus porcino como controles negativos. El 83.3 ± 12.3% (3...
2
artículo
Publicado 2011
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A real time RT-PCR assay was standardized and validated for detection of classical swine fever virus (CSFV). Tonsil tissue and lymph nodules that were positive (n=36) and negative (n= 30) to CSFV by immunofluorescence test (IF) were selected. The viral RNA extraction, the complementary DNA (cDNA) synthesis and real time RT- PCR were performed using commercial kits. Three sets of primers were tested for amplification of a conserved region (5’UTR) of panpestivirus and 5’UTR Chinese strain and the E2 glycoprotein region (E2 PPC) against reference strains of CSFV: Alfort/187, Brescia, and a vaccine Chinese strain as positive controls, and strains of the bovine viral diarrhea virus, border disease virus and porcine rotavirus as negative controls. The 83.3 ± 12.3% (30/36) of positive simples to CSFV by IF test was positive for virus isolation and the 96.7 ± 3.3% (29/30) of negative sampl...
3
artículo
Publicado 2023
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Objective: To optimize the internal quality control of real-time RT-PCR for the qualitative detection of SARS-CoV-2, using the Cq values of negative and positive controls. Material and method: Prospective-longitudinal study. The sample consisted of 143 Cq values for the negative aliquot and extraction controls, as well as for the positive control. The normal distribution of Cq values was analyzed using the Anderson-Darling (AD) test and randomness tests were applied. Control limits were calculated from 51 Cq values, and then, using control charts, to monitor 92 Cq values obtained from November 2020 to March 2021. Lot acceptance and Cpk indices were evaluated as optimization indicators. The calculations were made with the Minitab program. Results: The batches of Cq values were accepted and Cpk indices higher than 1.33 were obtained for the three types of cont...
4
artículo
Publicado 2015
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El objetivo del presente estudio fue determinar la expresión de péptidos antimicrobianos (á- y â-defensinas) en el epitelio intestinal de crías de alpaca de 1 a 6 semanas de edad y con enteropatías, mediante la cuantificación relativa de ARN mensajeros (ARNm) de las á (defa 8) y â (â def alp) defensinas. Se tomaron dos porciones de yeyuno de 2 cm de longitud de 29 crías de alpacas con signos de diarrea. Una porción se procesó para el análisis histopatológico utilizando la tinción Hematoxilina-Eosina para determinar el tipo de enteropatía. De la otra porción se obtuvo el ARNm total de los raspados yeyunales, que sirvió de molde para la síntesis de cDNA mediante transcripción reversa (RT), seguido de un PCR en Tiempo Real, para la amplificación y detección de las defensinas. La cuantificación relativa de ARNm se realizó mediante el método 2- ÄÄCt. Las crías enf...
5
artículo
Publicado 2015
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The aim of this study was to determine the expression of antimicrobial peptides (α- and β-defensins) in the intestinal epithelium of newborn alpaca (1 to 6 weeks of age) with enteropathies, using the relative quantification of messenger RNA (mRNA) of the α (Defa 8) and β (β def alp) defensins. Two portions of the jejunum (2 cm) were collected from 29 newborn alpaca with signs of diarrhea. One portion was processed for histological analysis using hematoxylin-eosin staining to determine the type of enteropathy. The second portion was used to obtain total mRNA from jejunal scrapings, which served as template for cDNA synthesis by reverse transcription (RT), followed by a Real-Time PCR for the amplification and detection of defensins. The relative quantification of mRNA was performed using 2-ΔΔCt method. The sick newborn showed an expression of α-defensin (Defa 8) corresponding to 18...
6
artículo
Publicado 2013
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The aim of this study was to evaluate the expression of proinflammatory cytokines of alpaca circulating leukocytes when confronting total extract of Clostridum perfringens (structural components and toxins). Whole blood was collected from the jugular vein of 5 female and 5 male adult alpacas. The leukocytes were separated using ammonium chloride to lyse erythrocytes followed by centrifugation and then cultured at a concentration of 500 000 cells/ml of MEM (Minimal Essential Medium) in culture plates of 12 wells. Simultaneously were challenged with total extract of C. perfringens grown in thioglycolate broth at concentrations of 400, 80, 16, 0.8 and 0.2 μg/ml and then quantifying by RT-PCR the expression of cytokines TNF∝, IL-1∝, IL-6 and IL-1ß at 1, 12 and 24 h of exposure. The results showed that low doses of clostridial extracts (0.2 to 0.8 mg/ml) are capable of inducing expressi...
7
artículo
Publicado 2013
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The aim of this study was to evaluate the expression of proinflammatory cytokines of alpaca circulating leukocytes when confronting total extract of Clostridum perfringens (structural components and toxins). Whole blood was collected from the jugular vein of 5 female and 5 male adult alpacas. The leukocytes were separated using ammonium chloride to lyse erythrocytes followed by centrifugation and then cultured at a concentration of 500 000 cells/ml of MEM (Minimal Essential Medium) in culture plates of 12 wells. Simultaneously were challenged with total extract of C. perfringens grown in thioglycolate broth at concentrations of 400, 80, 16, 0.8 and 0.2 μg/ml and then quantifying by RT-PCR the expression of cytokines TNF∝, IL-1∝, IL-6 and IL-1ß at 1, 12 and 24 h of exposure. The results showed that low doses of clostridial extracts (0.2 to 0.8 mg/ml) are capable of inducing expressi...
8
artículo
Publicado 2017
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El objetivo de este estudio fue determinar la expresión de genes del receptor TCRγδ (gamma y delta) en el epitelio yeyunal de 16 crías de alpaca aparentemente sanas, de 2 a 47 días de edad, mediante la cuantificación de ARN mensajero (ARNm) utilizando cebadores específicos. Se tomaron porciones de yeyuno (2 cm de longitud). El ARNm total de la mucosa de la porción media del yeyuno actuó como molde para la síntesis de ADN complementario mediante transcripción reversa (RT), seguida de un PCR-tiempo real para la amplificación y cuantificación de los ARNm de los polipéptidos que conforman las cadenas gamma y delta del TCR. Se utilizó el método 2-ΔΔCt para la cuantificación relativa de ARNm, teniendo como calibrador a dos crías neonatas que no habían consumido calostro. Las crías de 1, 2, 3 y ≥4 semanas de edad expresaron el gen gamma en 4.75, 6.78, 16.24 y 103.11 vece...
9
artículo
Publicado 2017
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The aim of this study was to determine the expression of TCRγδ receptor genes (gamma and delta) in the jejunal epithelium of 16 apparently healthy baby alpacas (2 to 47 days of age), by quantifying messenger RNA (mRNA) using specific primers. Jejunumsamples (2 cm long) were collected. Total mRNA of the medium portion of the jejunum acted as template for complementary DNA synthesis by reverse transcription (RT), followed by real-time PCR for the amplification and quantification of the mRNAs of the polypeptides that make up the gamma and delta chains of the TCR. The 2-ΔΔCt method was used for the relative mRNA quantification, using as calibrator two neonatal alpacas that had not consumed colostrum. The alpacas of 1, 2, 3 and >4 weeks of age expressed the gamma gene at 4.75, 6.78, 16.24 and 103.11 folds as expressed by the calibrator animals, respectively, and the delta gene was expr...
10
artículo
Publicado 2012
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The objective of this study was to standardize and validate the real-time RT-PCR technique for diagnosis of Infectious Pancreatic Necrosis virus (IPNV) in rainbow trout (Oncorhynchus mykiss) from Central Sierra of Peru. Samples of kidney and spleen (n=61) of rainbow trout with apparent clinical signs of IPN and from rainbow trout apparently healthy (n=60) were collected from two fish farms located in the central sierra. Before standardization of the real-time RT-PCR technique, the kidney and spleen samples of 121 rainbow trout were tested for antigen of IPNV by indirect immunofluorescent (IIF) test. In addition, kidney and spleen samples of 10 rainbow trout negative to IPNV by IIF were inoculated with a serial dilution of IPNV strain (from initial concentration of 103 PFU/ml). The RNA extraction of the 121 samples and of the 10 samples inoculated with IPNV strain, the cDNA and the real-t...
11
artículo
Publicado 2012
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The objective of this study was to standardize and validate the real-time RT-PCR technique for diagnosis of Infectious Pancreatic Necrosis virus (IPNV) in rainbow trout (Oncorhynchus mykiss) from Central Sierra of Peru. Samples of kidney and spleen (n=61) of rainbow trout with apparent clinical signs of IPN and from rainbow trout apparently healthy (n=60) were collected from two fish farms located in the central sierra. Before standardization of the real-time RT-PCR technique, the kidney and spleen samples of 121 rainbow trout were tested for antigen of IPNV by indirect immunofluorescent (IIF) test. In addition, kidney and spleen samples of 10 rainbow trout negative to IPNV by IIF were inoculated with a serial dilution of IPNV strain (from initial concentration of 103 PFU/ml). The RNA extraction of the 121 samples and of the 10 samples inoculated with IPNV strain, the cDNA and the real-t...
12
artículo
Publicado 2018
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El estudio tuvo por objetivo desarrollar una plataforma molecular para la cuantificación del virus de la enfermedad de Newcastle (NDV) a partir de un sistema de cultivo en huevos embrionados SPF. Primero se evaluaron cuatro pares de cebadores que amplifican diferentes regiones del genoma viral de NDV que codifican para la proteína de nucleocápside (NP), proteína matriz (M), proteína fusión (F) y la ARN polimerasa ARN dependiente (L), con la finalidad de seleccionar el más conservado a partir del cual se desarrolló una plataforma molecular basada en la transcripción reversa - reacción en cadena de polimerasa convencional (RT-PCRc) en dos pasos para la detección del NDV. Posteriormente, esta fue llevada a transcripción reversa - reacción en cadena de polimerasa en tiempo real (RT-qPCR) para la cuantificación de NDV producido a partir de un sistema de huevos embrionados. Media...
13
artículo
Publicado 2017
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El objetivo del trabajo fue evaluar los niveles de expresión relativa de los genes de las citoquinas IL-10 y TGF-β en la mucosa intestinal de alpacas de 2 a 47 días de edad, clínicamente sanas. Se formaron tres grupos etarios (seis crías por grupo) conformados por alpacas de 2 a 8 días (grupo 1), 10 a 21 días (grupo 2) y 26 a 47 días (grupo 3), sin distinción de sexo o raza. Se tomó la porción media del yeyuno de cada animal, se extrajo el ARN total y se empleó la técnica RT-PCR en tiempo-real con cebadores específicos para las citoquinas en estudio. La expresión relativa de ARNm de IL10 y TGF-β fue determinada por el método comparativo 2-ΔΔCt usando como calibrador el yeyuno de tres fetos de 11 meses de gestación y como gen endógeno el gliceraldehído-3-fosfato deshidrogenasa (GAPDH). La expresión promedio de ARNm de IL-10 fue de 7.21±1.02 (grupo 1), 13.53±1.26 (...
14
artículo
Publicado 2017
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The aim of the present study was to evaluate the relative expression levels of cytokines IL-10 and TGF-β in the intestinal mucosa of clinically healthy alpacas from 2 to 47 days old. The animals were classified in three age groups (six animals per group): 2-8 days old (group 1), 10-21days old (group 2) and 26-47-days old (group 3), regardless sex or breed. The midportion of jejunum of each animal was collected; total RNA was extracted and then analyzed by real-time RT-PCR technique using IL-10 and TGF-β specific primers. The relative mRNA expression analysis was done using the comparative 2-ΔΔCt method. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as housekeeping gene and jejunum of three 11-month-old fetuses were used as calibrators. The results showed that IL-10 mRNA expression levels were 7.21±1.02 (group 1), 13.53±1.26 (group 2), and 18.77±1.48 (group 3) times as ...
15
artículo
Publicado 2011
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El objetivo del presente estudio fue determinar la presencia genómica de ? - y ?- defensinas (Defa 8 y??-def alp) y sus niveles de expresión como ARN mensajeros, en muestras de mucosa intestinal de crías alpaca. Se colectaron muestras de yeyuno y sangre de 30 animales de 0 a 45 días de edad. Se extrajo el ADN genómico y los ARN mensajeros para el análisis por PCR y RT-PCR tiempo real, respectivamente. La prueba de PCR convencional determinó que todas las muestras amplificaron un segmento genómico de entre 800 y 900 pb correspondiente a una Defa 8, y el 90% (27/30) de las muestras amplificó un segmento esperado de 300 a 350 pb para ?-def alp y dos bandas adicionales de 800 a 900 pb, evidenciados en la electroforesis en gel de agarosa. La detección de ARN mensajeros por RT-PCR tiempo real se realizó en base al análisis de las curvas de amplificación (Ct) y curvas de disociaci...
16
artículo
Publicado 2011
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The aim of this study was to determine the presence of genomic ?-and ? -defensins (bovine enteric beta defensin – ?-def alp – and Defa 8 respectively) and their levels of expression as messenger RNA in samples of intestinal mucosa of newborn alpacas. Samples of jejunum of 30 animals from 0 to 45 days of age were collected. Genomic DNA and mRNA were extracted for PCR analysis and real-time RT-PCR respectively. All samples amplified a single DNA segment (between 800 and 900 pb) by PCR which corresponds to the alpha defensin 8 (defa 8), while 90% (27/30) amplified a segment expected 300-350 pb for ?-def alp and two additional bands of 800-900 pb evidenced by agarose gel electrophoresis. The detection of mRNAs by real time RT-PCR was performed based on Cycle threshold (Ct) analysis and Melting temperature (Tm) of the amplified products. The Cts for defa 8 products were found between 25.1...
17
artículo
Publicado 2013
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The aim of this study was to determine by real-time RT-PCR the presence and relative levels of tumor necrosis factor alpha (TNF-a) and interleukin 1 alpha (IL-1a) mRNA expression in intestinal mucosa of healthy and with enteropathy young alpacas (Vicugna pacos) from Cusco, Peru. A total of 71 samples of jejunum from 0 to 45 days old healthy (n=38) and diseased (n=33) alpacas were processed. Total RNA was extracted from samples and to the synthesis of DNA complementary strand (cDNA) by reverse transcription (RT) was done. Subsequently, conventional PCR and real time RT-PCR was conducted. The results showed that 98.6% (70/71) of the samples amplified a specific product of 251 pb for TNF-a and 100% (71/71) of the samples amplified a specific product of 172 pb for IL-1a, evidenced in the agarose gel during electrophoresis. Analysis of dissociation curves determined that: a) the products ampl...
18
artículo
Publicado 2013
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The aim of this study was to determine by real-time RT-PCR the presence and relative levels of tumor necrosis factor alpha (TNF-a) and interleukin 1 alpha (IL-1a) mRNA expression in intestinal mucosa of healthy and with enteropathy young alpacas (Vicugna pacos) from Cusco, Peru. A total of 71 samples of jejunum from 0 to 45 days old healthy (n=38) and diseased (n=33) alpacas were processed. Total RNA was extracted from samples and to the synthesis of DNA complementary strand (cDNA) by reverse transcription (RT) was done. Subsequently, conventional PCR and real time RT-PCR was conducted. The results showed that 98.6% (70/71) of the samples amplified a specific product of 251 pb for TNF-a and 100% (71/71) of the samples amplified a specific product of 172 pb for IL-1a, evidenced in the agarose gel during electrophoresis. Analysis of dissociation curves determined that: a) the products ampl...
19
artículo
Publicado 2014
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The aim of the study was to determine the relative in vitro ARNm expression levels of IL-2 and IL-10 in peripheral blood leukocytes by real time RT-PCR and relative quantification using the 2-ΔΔCt method in presence of clostridial antigens and GAPDH as an endogenous control. Whole blood was collected from 10 adult alpacas. Samples were centrifuged to obtain leukocytes using the Ficoll reagent, purified with ammonium chloride and cultured in 24-well plates at a concentration of 500 000 live cells/ml minimal essential medium (MEM) with clostridial extract suspensions at concentrations of 400, 16, 0.8 and 0.2 µg/ml. Subsequently, the RT-qPCR test was performed. IL-2 kinetic expression patterns were inversely proportional to the clostridial antigen dose in the three incubation times (p>0.05), unlike IL-10 which its kinetic expression patterns were variable, reaching its maximum at 12 h...
20
artículo
Publicado 2014
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The aim of the study was to determine the relative in vitro ARNm expression levels of IL-2 and IL-10 in peripheral blood leukocytes by real time RT-PCR and relative quantification using the 2-ΔΔCt method in presence of clostridial antigens and GAPDH as an endogenous control. Whole blood was collected from 10 adult alpacas. Samples were centrifuged to obtain leukocytes using the Ficoll reagent, purified with ammonium chloride and cultured in 24-well plates at a concentration of 500 000 live cells/ml minimal essential medium (MEM) with clostridial extract suspensions at concentrations of 400, 16, 0.8 and 0.2 µg/ml. Subsequently, the RT-qPCR test was performed. IL-2 kinetic expression patterns were inversely proportional to the clostridial antigen dose in the three incubation times (p>0.05), unlike IL-10 which its kinetic expression patterns were variable, reaching its maximum at 12 h...
