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Registro completo
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Biblioteca (s) : |
INIA Las Brujas. |
Fecha : |
04/10/2014 |
Actualizado : |
06/06/2023 |
Tipo de producción científica : |
Artículos en Revistas Indexadas Internacionales |
Autor : |
UMAÑA, R.; PRITSCH, C.; ARBIZA, J.R.; RIVAS, F.; PAGLIANO, G. |
Afiliación : |
CARLOS FERNANDO RIVAS GRELA, INIA (Instituto Nacional de Investigación Agropecuaria), Uruguay. |
Título : |
Evaluation of four viroid rna extraction methods for the molecular diagnosis of cevd in citrus lemon using rt-pcr, dot blot and northern blot. [Evaluación de cuatro métodos de extracción de ARN viroide para el diagnóstico molecular de CEVd en Citrus limon mediante RT-PCR, Dot blot y Northern blot.]. |
Fecha de publicación : |
2013 |
Fuente / Imprenta : |
Biotecnologia Aplicada, 2013, v.30, no.2, p.125-130. |
Descripción física : |
2-s2.0-84884171392 |
Serie : |
1027-2852 |
ISSN : |
1027-2852 |
Idioma : |
Inglés |
Contenido : |
ABSTRACT.
An efficient method for RNA extraction that leads to RNA high yield and purity is a technical issue relevant for development and optimization of molecular diagnostic methods aimed to detect viroid infections in citrus varieties. Residual contaminants may affect RNA detection depending on the molecular diagnosis approaches. This condition can be evaluated through RNA absorption spectrum analysis. Functionally, it is assessed through observation of RTPCR amplification products and Northern blot and Dot-blot signal intensities, displaying levels of analytical response/ sensitivity. Four RNA extraction methods were evaluated to determine their effects on the capacity to detect viroid CEVd presence/absence in Citrus limon through four molecular diagnostic approaches: 1) conventional viroid extraction (CVE); 2) phenol/guanidine thiocyanate (PGT), 3) SDS/potassium acetate (SPA); and 4) formaldehyde/ SSC (FS). Phloem tissue quantifications showed values between 7500 ng/μL and 1200 ng/μL and ranged 1.3-2.0 OD260/280. Evaluations through RT-PCR showed the expected amplifications of the entire CEVd genome, but erratic scenarios still remained. Non-radioactive probe hybridization techniques revealed high intensity signals (132 RU) for infected tissue, by using the CVE method, and a positivity cut-off for the presence of infection was established (78 RU). Nevertheless, molecular hybridization tools can jeopardize the diagnosis due to the thoroughness of the protocol and the RNA template conditions. The diagnostic ability of the association of Northern blot with CVE viroid extraction
analyses as starting point was evidenced for successful detection, among the molecular methods tested.
RESUMEN.Evaluación de cuatro métodos de extracción de ARN viroide para el diagnóstico molecular de CEVd en Citrus limon mediante RT-PCR, Dot blot y Northern blot. En el desarrollo de metodologías moleculares diagnósticas en variedades cítricas propensas a infección viroide, se precisa la extracción efi ciente de ARN, siguiendo criterios de concentración y pureza. Los contaminantes remanentes pueden afectar la detección según la herramienta molecular escogida. Esta condición se analiza a partir del espectro de absorción del ARN; mediante la reacción en cadena de la polimerasa con transcriptasa inversa (RT-PCR) y por los niveles de intensidad de las señales de Northern blot y Dot blot, en términos de respuesta analítica/sensibilidad. Se evaluaron cuatro métodos de extracción de ARN, por sus
efectos sobre la detección de presencia/ausencia del Citrus Exocortis Viroid (CEVd) en Citrus limon mediante análisis moleculares diagnósticos: 1) extracción viroide convencional (EVC); 2) fenol/tiocianato de guanidina (FTG); 3) SDS/ acetato de potasio (SAP); y 4) formaldehído/SSC (FS). Los valores del tejido de fl oema estuvieron entre 7500 ng/μL y 1200 ng/μL y los rangos entre 1.3 y 2.0 DO260/280. La evaluación por aproximaciones de la RT-PCR reflejó las amplifi caciones esperadas del genoma completo del CEVd; sin embargo, aún se discuten los escenarios erráticos.
Las hibridaciones no radiactivas revelaron señales de alta intensidad (132 UR) para el tejido infectado, según el método de EVC, y la defi nición del límite de positividad para la presencia de infección (78 UR). Las herramientas basadas en hibridaciones moleculares interfi eren en el diagnóstico, por la rigurosidad del protocolo y las condiciones del ARN molde. La extracción viroide como punto de partida de una detección exitosa y los métodos moleculares ensayados, mostraron las posibilidades diagnósticas de la asociación de Northern blot con la EVC. MenosABSTRACT.
An efficient method for RNA extraction that leads to RNA high yield and purity is a technical issue relevant for development and optimization of molecular diagnostic methods aimed to detect viroid infections in citrus varieties. Residual contaminants may affect RNA detection depending on the molecular diagnosis approaches. This condition can be evaluated through RNA absorption spectrum analysis. Functionally, it is assessed through observation of RTPCR amplification products and Northern blot and Dot-blot signal intensities, displaying levels of analytical response/ sensitivity. Four RNA extraction methods were evaluated to determine their effects on the capacity to detect viroid CEVd presence/absence in Citrus limon through four molecular diagnostic approaches: 1) conventional viroid extraction (CVE); 2) phenol/guanidine thiocyanate (PGT), 3) SDS/potassium acetate (SPA); and 4) formaldehyde/ SSC (FS). Phloem tissue quantifications showed values between 7500 ng/μL and 1200 ng/μL and ranged 1.3-2.0 OD260/280. Evaluations through RT-PCR showed the expected amplifications of the entire CEVd genome, but erratic scenarios still remained. Non-radioactive probe hybridization techniques revealed high intensity signals (132 RU) for infected tissue, by using the CVE method, and a positivity cut-off for the presence of infection was established (78 RU). Nevertheless, molecular hybridization tools can jeopardize the diagnosis due to the thoroughness of the protocol ... Presentar Todo |
Palabras claves : |
CEVd. |
Thesagro : |
CITRUS; DETECCIÓN DE VIROIDES DE CITRUS; DIAGNOSTICO; ENFERMEDADES DE LOS CITRUS; TECNICAS; VIROSIS. |
Asunto categoría : |
-- H20 Enfermedades de las plantas |
URL : |
http://www.ainfo.inia.uy/digital/bitstream/item/3438/1/Rivas-F.-2013.-Biotec.Aplicada-v.302-p.125-130.pdf
http://www.ainfo.inia.uy/digital/bitstream/item/3439/1/Rivas-F.-2013.-Biotec.Aplicada-v.302-p.131-136.pdf
http://www.medigraphic.com/pdfs/biotecapl/ba-2013/ba132g.pdf
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Marc : |
LEADER 04714naa a2200289 a 4500 001 1050903 005 2023-06-06 008 2013 bl uuuu u00u1 u #d 022 $a1027-2852 100 1 $aUMAÑA, R. 245 $aEvaluation of four viroid rna extraction methods for the molecular diagnosis of cevd in citrus lemon using rt-pcr, dot blot and northern blot. [Evaluación de cuatro métodos de extracción de ARN viroide para el diagnóstico molecular de CEVd en Citrus limon mediante RT-PCR, Dot blot y Northern blot.].$h[electronic resource] 260 $c2013 300 $c2-s2.0-84884171392 490 $a1027-2852 520 $aABSTRACT. An efficient method for RNA extraction that leads to RNA high yield and purity is a technical issue relevant for development and optimization of molecular diagnostic methods aimed to detect viroid infections in citrus varieties. Residual contaminants may affect RNA detection depending on the molecular diagnosis approaches. This condition can be evaluated through RNA absorption spectrum analysis. Functionally, it is assessed through observation of RTPCR amplification products and Northern blot and Dot-blot signal intensities, displaying levels of analytical response/ sensitivity. Four RNA extraction methods were evaluated to determine their effects on the capacity to detect viroid CEVd presence/absence in Citrus limon through four molecular diagnostic approaches: 1) conventional viroid extraction (CVE); 2) phenol/guanidine thiocyanate (PGT), 3) SDS/potassium acetate (SPA); and 4) formaldehyde/ SSC (FS). Phloem tissue quantifications showed values between 7500 ng/μL and 1200 ng/μL and ranged 1.3-2.0 OD260/280. Evaluations through RT-PCR showed the expected amplifications of the entire CEVd genome, but erratic scenarios still remained. Non-radioactive probe hybridization techniques revealed high intensity signals (132 RU) for infected tissue, by using the CVE method, and a positivity cut-off for the presence of infection was established (78 RU). Nevertheless, molecular hybridization tools can jeopardize the diagnosis due to the thoroughness of the protocol and the RNA template conditions. The diagnostic ability of the association of Northern blot with CVE viroid extraction analyses as starting point was evidenced for successful detection, among the molecular methods tested. RESUMEN.Evaluación de cuatro métodos de extracción de ARN viroide para el diagnóstico molecular de CEVd en Citrus limon mediante RT-PCR, Dot blot y Northern blot. En el desarrollo de metodologías moleculares diagnósticas en variedades cítricas propensas a infección viroide, se precisa la extracción efi ciente de ARN, siguiendo criterios de concentración y pureza. Los contaminantes remanentes pueden afectar la detección según la herramienta molecular escogida. Esta condición se analiza a partir del espectro de absorción del ARN; mediante la reacción en cadena de la polimerasa con transcriptasa inversa (RT-PCR) y por los niveles de intensidad de las señales de Northern blot y Dot blot, en términos de respuesta analítica/sensibilidad. Se evaluaron cuatro métodos de extracción de ARN, por sus efectos sobre la detección de presencia/ausencia del Citrus Exocortis Viroid (CEVd) en Citrus limon mediante análisis moleculares diagnósticos: 1) extracción viroide convencional (EVC); 2) fenol/tiocianato de guanidina (FTG); 3) SDS/ acetato de potasio (SAP); y 4) formaldehído/SSC (FS). Los valores del tejido de fl oema estuvieron entre 7500 ng/μL y 1200 ng/μL y los rangos entre 1.3 y 2.0 DO260/280. La evaluación por aproximaciones de la RT-PCR reflejó las amplifi caciones esperadas del genoma completo del CEVd; sin embargo, aún se discuten los escenarios erráticos. Las hibridaciones no radiactivas revelaron señales de alta intensidad (132 UR) para el tejido infectado, según el método de EVC, y la defi nición del límite de positividad para la presencia de infección (78 UR). Las herramientas basadas en hibridaciones moleculares interfi eren en el diagnóstico, por la rigurosidad del protocolo y las condiciones del ARN molde. La extracción viroide como punto de partida de una detección exitosa y los métodos moleculares ensayados, mostraron las posibilidades diagnósticas de la asociación de Northern blot con la EVC. 650 $aCITRUS 650 $aDETECCIÓN DE VIROIDES DE CITRUS 650 $aDIAGNOSTICO 650 $aENFERMEDADES DE LOS CITRUS 650 $aTECNICAS 650 $aVIROSIS 653 $aCEVd 700 1 $aPRITSCH, C. 700 1 $aARBIZA, J.R. 700 1 $aRIVAS, F. 700 1 $aPAGLIANO, G. 773 $tBiotecnologia Aplicada, 2013$gv.30, no.2, p.125-130.
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INIA Las Brujas (LB) |
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Registro completo
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Biblioteca (s) : |
INIA Las Brujas. |
Fecha actual : |
06/10/2014 |
Actualizado : |
10/02/2020 |
Tipo de producción científica : |
Artículos en Revistas Indexadas Internacionales |
Circulación / Nivel : |
A - 2 |
Autor : |
SIMEONE, R.; MISZTAL, I.; AGUILAR, I.; LEGARRA, A. |
Afiliación : |
IGNACIO AGUILAR GARCIA, Instituto Nacional de Investigación Agropecuaria (INIA), Uruguay. |
Título : |
Evaluation of the utility of diagonal elements of the genomic relationship matrix as a diagnostic tool to detect mislabelled genotyped animals in a broiler chicken population. |
Fecha de publicación : |
2011 |
Fuente / Imprenta : |
Journal of Animal Breeding and Genetics, 2011, v.128, no.5, p.386-393. |
ISSN : |
0931-2668 |
DOI : |
http://dx.doi.org/10.1111/j.1439-0388.2011.00926.x |
Idioma : |
Inglés |
Contenido : |
ABSTRACT,
This study explored distributions of diagonal elements of genomic relationship matrix (G), evaluated the utility of G as a diagnostic tool to detect mislabelled animals in a genomic dataset and evaluated the effect of mislabelled animals on the accuracy of genomic evaluation. Populations of 10000 animals were simulated with 60000 SNP varying in allele frequency at each locus between 0.02 and 0.98. Diagonal elements of G were distributed with a single peak (mean=1.00±0.03) and ranged from 0.84 through 1.36. Mixed populations were also simulated: 7000 animals with frequencies of second alleles ranging from 0.02 through 0.98 were combined with 1750 or 7000 animals with frequencies of second alleles ranging from 0.0 through 1.0. The resulting distributions of diagonal elements of G were bimodal. Body weight at 6weeks was provided by Cobb-Vantress for broiler chickens, of which 3285 were genotyped for 57636 SNP. Analysis used a combined genomic and pedigree relationship matrix; G was scaled using current allele frequencies. The distribution of diagonal elements was multimodal and ranged from 0.54 to 3.23. Animals with diagonal elements >1.5 were identified as coming from another chicken line or as having low call rates. Removal of mislabelled animals increased accuracy by 0.01. For the studied type of population, diagonal elements of G may be a useful tool to help identify mislabelled animals or secondary populations.
© 2011 Blackwell Verlag GmbH. |
Thesagro : |
GENOTIPOS; MEJORAMIENTO GENÉTICO ANIMAL; MODELOS DE SIMULACIÓN; POLLO; SELECCIÓN DE GENOTIPOS. |
Asunto categoría : |
L10 Genética y mejoramiento animal |
Marc : |
LEADER 02325naa a2200241 a 4500 001 1050930 005 2020-02-10 008 2011 bl uuuu u00u1 u #d 022 $a0931-2668 024 7 $ahttp://dx.doi.org/10.1111/j.1439-0388.2011.00926.x$2DOI 100 1 $aSIMEONE, R. 245 $aEvaluation of the utility of diagonal elements of the genomic relationship matrix as a diagnostic tool to detect mislabelled genotyped animals in a broiler chicken population.$h[electronic resource] 260 $c2011 520 $aABSTRACT, This study explored distributions of diagonal elements of genomic relationship matrix (G), evaluated the utility of G as a diagnostic tool to detect mislabelled animals in a genomic dataset and evaluated the effect of mislabelled animals on the accuracy of genomic evaluation. Populations of 10000 animals were simulated with 60000 SNP varying in allele frequency at each locus between 0.02 and 0.98. Diagonal elements of G were distributed with a single peak (mean=1.00±0.03) and ranged from 0.84 through 1.36. Mixed populations were also simulated: 7000 animals with frequencies of second alleles ranging from 0.02 through 0.98 were combined with 1750 or 7000 animals with frequencies of second alleles ranging from 0.0 through 1.0. The resulting distributions of diagonal elements of G were bimodal. Body weight at 6weeks was provided by Cobb-Vantress for broiler chickens, of which 3285 were genotyped for 57636 SNP. Analysis used a combined genomic and pedigree relationship matrix; G was scaled using current allele frequencies. The distribution of diagonal elements was multimodal and ranged from 0.54 to 3.23. Animals with diagonal elements >1.5 were identified as coming from another chicken line or as having low call rates. Removal of mislabelled animals increased accuracy by 0.01. For the studied type of population, diagonal elements of G may be a useful tool to help identify mislabelled animals or secondary populations. © 2011 Blackwell Verlag GmbH. 650 $aGENOTIPOS 650 $aMEJORAMIENTO GENÉTICO ANIMAL 650 $aMODELOS DE SIMULACIÓN 650 $aPOLLO 650 $aSELECCIÓN DE GENOTIPOS 700 1 $aMISZTAL, I. 700 1 $aAGUILAR, I. 700 1 $aLEGARRA, A. 773 $tJournal of Animal Breeding and Genetics, 2011$gv.128, no.5, p.386-393.
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