Comparison of two affordable DNA extraction methods for molecular detection of Salmonella isolates from broiler farm’s boot swabs
Autor(a) principal: | |
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Data de Publicação: | 2023 |
Outros Autores: | , , , , , |
Tipo de documento: | Artigo |
Idioma: | eng |
Título da fonte: | Research, Society and Development |
Texto Completo: | https://rsdjournal.org/index.php/rsd/article/view/39618 |
Resumo: | There are many extraction methods available to purify bacterial DNA. PCR efficiency can vary depending on the quantity and quality of the template DNA used. This study evaluated the efficiency, quality, cost and rapidness of two in-house protocols, silica particles and Chelex-100 resin, for the extraction of twenty Salmonella isolates from boot swabs. The aim of this experiment was to compare the extraction methods for Salmonella spp. detection. DNA extraction was performed for each method and subjected to real-time PCR amplification. The amounts and integrity of DNA were determined using spectrophotometry and agarose gel electrophoresis, and the efficiency measured with real-time PCR. Limit of Detection (LOD) was determined with serial dilutions of a S. Typhimurium reference strain resulting in linear regression coefficients of R 2=0.992 (efficiency 119.31) for silica and R 2=0.958 (efficiency 93.33) for Chelex, with LOD of 10 -4 for both. Silica particles method resulted in higher DNA yield, 85.01 ± 59.11 compared to 50.74 ± 12.95 and 260/280 ratio, 1.77 ± 0.02 versus 1.63 ± 0.13. DNA integrity was superior using silica, gel showed a unique band higher than 2.000 bp, while Chelex-100 was imperceptive or degraded. Regarding PCR results, the mean quantification cycle (Cq) for silica was 17.08 ± 0.73 and Chelex-100, 17.64 ±0.56 (suggesting lower DNA values). Results showed that both methods were effective for the DNA extraction of Salmonella, once PCR resulted positive for all samples, efficiency was higher for silica. Chelex-100 resin was performed in less time at a lower cost. |
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Comparison of two affordable DNA extraction methods for molecular detection of Salmonella isolates from broiler farm’s boot swabsComparación de dos métodos asequibles de extracción de ADN para la detección molecular de cepas de Salmonella aisladas de calzas de celulosa de granjas de pollos de engordeComparação de dois métodos de purificação de DNA de baixo custo para a detecção molecular de Salmonella em isolados de propés de aviáriosPCR em tempo realSílicaChelex-100Salmonella.PCR en tiempo realSílicaChelex-100Salmonella.Real-time PCRSilica particlesChelex-100 resinSalmonella.There are many extraction methods available to purify bacterial DNA. PCR efficiency can vary depending on the quantity and quality of the template DNA used. This study evaluated the efficiency, quality, cost and rapidness of two in-house protocols, silica particles and Chelex-100 resin, for the extraction of twenty Salmonella isolates from boot swabs. The aim of this experiment was to compare the extraction methods for Salmonella spp. detection. DNA extraction was performed for each method and subjected to real-time PCR amplification. The amounts and integrity of DNA were determined using spectrophotometry and agarose gel electrophoresis, and the efficiency measured with real-time PCR. Limit of Detection (LOD) was determined with serial dilutions of a S. Typhimurium reference strain resulting in linear regression coefficients of R 2=0.992 (efficiency 119.31) for silica and R 2=0.958 (efficiency 93.33) for Chelex, with LOD of 10 -4 for both. Silica particles method resulted in higher DNA yield, 85.01 ± 59.11 compared to 50.74 ± 12.95 and 260/280 ratio, 1.77 ± 0.02 versus 1.63 ± 0.13. DNA integrity was superior using silica, gel showed a unique band higher than 2.000 bp, while Chelex-100 was imperceptive or degraded. Regarding PCR results, the mean quantification cycle (Cq) for silica was 17.08 ± 0.73 and Chelex-100, 17.64 ±0.56 (suggesting lower DNA values). Results showed that both methods were effective for the DNA extraction of Salmonella, once PCR resulted positive for all samples, efficiency was higher for silica. Chelex-100 resin was performed in less time at a lower cost.Existen muchos métodos para purificar ADN bacteriano. La eficiencia de la PCR puede variar dependiendo de la cantidad y cualidad de ADN utilizada. Este estudio evaluó la eficiencia, calidad, costo y rapidez de dos protocolos, sílica y Chelex-100 para la extracción de veinte aislados de Salmonella provenientes de calzas de celulosa. El objetivo del trabajo fue comparar los métodos de extracción para la detección de especies de Salmonella. ADN fue extraído y sometido a PCR en tiempo real. La cantidad e integridad fue determinada por espectrofotometría y electroforesis en gel de agarosa y eficiencia por qPCR. Para calcular el límite de detección (LOD), una cepa de referencia S. Typhimurium fue diluida en serie resultando en coeficientes de regresión lineal de R 2=0,992 (eficiência 119,31) y R 2=0,958 (eficiência 93,33) para sílica y Chelex, con el mismo LOD de 10 -4. La sílica resultó en mayor cantidad de ADN, 85,01 ± 59,11 comparada a 50,74 ± 12,95 y para la razón 260/280 1,77 ± 0,02 versus 1,63 ± 0,13. La integridad de la sílica pareció mejor en el gel, con una banda superior a 2.000 pb, ADN de Chelex-100 pareció imperceptible o degradado. La media del ciclo de cuantificación (Cq) de la sílica fue 17,08 ± 0,73 y del Chelex-100, 17,64 ±0,56 (sugiriendo menor concentración de ADN). Los resultados demostraron que los dos métodos fueron capaces de detectar Salmonella, una vez que la PCR fue positiva para todas las muestras, la eficiencia resultó superior para sílica. Chelex-100 fue más rápido y asequible.Existem muitos métodos de extração para purificar DNA bacteriano. A eficiência da PCR pode variar dependendo da quantidade e qualidade do DNA alvo utilizado. Este estudo avaliou a eficiência, qualidade, custo e rapidez de dois protocolos, sílica e Chelex-100, para a extração de vinte isolados de Salmonella oriundos de propés. O objetivo do experimento foi comparar os métodos de extração para detecção de espécies de Salmonella. O DNA foi extraído e submetido à PCR em tempo-real. Determinou-se a quantidade e integridade do DNA por espectrofotometria e eletroforese em gel de agarose e eficiência por qPCR. Para calcular o limite de detecção (LOD) uma cepa de referência S. Typhimurium foi diluída em série resultando nos coeficientes de regressão linear de R 2=0,992 (eficiencia 119,31) e R 2=0,958 (eficiencia 93,33) para sílica e Chelex, com LOD de 10 -4 para ambos. A Sílica rendeu mais DNA, 85,01 ± 59,11 comparado a 50,74 ± 12,95 e a razão 260/280 foi 1,77 ± 0,02 versus 1,63 ± 0,13. A sílica apresentou melhor integridade, gel com banda superior a 2.000 pb, enquanto o DNA do Chelex-100 pareceu imperceptível ou degradado. Com relação à PCR, a média do ciclo de quantificação (Cq) para sílica foi: 17,08 ± 0,73 e Chelex-100: 17,64 ±0,56 (sugerindo menor concentração de DNA). Os resultados mostraram que os métodos foram efetivos para a extração de Salmonella, uma vez que a PCR resultou positiva para todas as amostras, com eficiência superior para a sílica. O método Chelex-100 foi mais rápido e econômico.Research, Society and Development2023-01-12info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionapplication/pdfhttps://rsdjournal.org/index.php/rsd/article/view/3961810.33448/rsd-v12i1.39618Research, Society and Development; Vol. 12 No. 1; e28312139618Research, Society and Development; Vol. 12 Núm. 1; e28312139618Research, Society and Development; v. 12 n. 1; e283121396182525-3409reponame:Research, Society and Developmentinstname:Universidade Federal de Itajubá (UNIFEI)instacron:UNIFEIenghttps://rsdjournal.org/index.php/rsd/article/view/39618/32620Copyright (c) 2023 Julia Iracema Moura Pacheco; Ketlen Beatriz Alves dos Anjos; Isabela Vaz Silva; Rodrigo Gibrail Okar; Sonia Maria Biesdorf Dorneles Rodrigues; Aniê Ieda Francabandiera; Maria Constanza Rodriguezhttps://creativecommons.org/licenses/by/4.0info:eu-repo/semantics/openAccessPacheco, Julia Iracema Moura Anjos, Ketlen Beatriz Alves dos Silva, Isabela Vaz Okar, Rodrigo Gibrail Rodrigues, Sonia Maria Biesdorf Dorneles Francabandiera, Aniê IedaRodriguez, Maria Constanza2023-01-13T10:30:42Zoai:ojs.pkp.sfu.ca:article/39618Revistahttps://rsdjournal.org/index.php/rsd/indexPUBhttps://rsdjournal.org/index.php/rsd/oairsd.articles@gmail.com2525-34092525-3409opendoar:2023-01-13T10:30:42Research, Society and Development - Universidade Federal de Itajubá (UNIFEI)false |
dc.title.none.fl_str_mv |
Comparison of two affordable DNA extraction methods for molecular detection of Salmonella isolates from broiler farm’s boot swabs Comparación de dos métodos asequibles de extracción de ADN para la detección molecular de cepas de Salmonella aisladas de calzas de celulosa de granjas de pollos de engorde Comparação de dois métodos de purificação de DNA de baixo custo para a detecção molecular de Salmonella em isolados de propés de aviários |
title |
Comparison of two affordable DNA extraction methods for molecular detection of Salmonella isolates from broiler farm’s boot swabs |
spellingShingle |
Comparison of two affordable DNA extraction methods for molecular detection of Salmonella isolates from broiler farm’s boot swabs Pacheco, Julia Iracema Moura PCR em tempo real Sílica Chelex-100 Salmonella. PCR en tiempo real Sílica Chelex-100 Salmonella. Real-time PCR Silica particles Chelex-100 resin Salmonella. |
title_short |
Comparison of two affordable DNA extraction methods for molecular detection of Salmonella isolates from broiler farm’s boot swabs |
title_full |
Comparison of two affordable DNA extraction methods for molecular detection of Salmonella isolates from broiler farm’s boot swabs |
title_fullStr |
Comparison of two affordable DNA extraction methods for molecular detection of Salmonella isolates from broiler farm’s boot swabs |
title_full_unstemmed |
Comparison of two affordable DNA extraction methods for molecular detection of Salmonella isolates from broiler farm’s boot swabs |
title_sort |
Comparison of two affordable DNA extraction methods for molecular detection of Salmonella isolates from broiler farm’s boot swabs |
author |
Pacheco, Julia Iracema Moura |
author_facet |
Pacheco, Julia Iracema Moura Anjos, Ketlen Beatriz Alves dos Silva, Isabela Vaz Okar, Rodrigo Gibrail Rodrigues, Sonia Maria Biesdorf Dorneles Francabandiera, Aniê Ieda Rodriguez, Maria Constanza |
author_role |
author |
author2 |
Anjos, Ketlen Beatriz Alves dos Silva, Isabela Vaz Okar, Rodrigo Gibrail Rodrigues, Sonia Maria Biesdorf Dorneles Francabandiera, Aniê Ieda Rodriguez, Maria Constanza |
author2_role |
author author author author author author |
dc.contributor.author.fl_str_mv |
Pacheco, Julia Iracema Moura Anjos, Ketlen Beatriz Alves dos Silva, Isabela Vaz Okar, Rodrigo Gibrail Rodrigues, Sonia Maria Biesdorf Dorneles Francabandiera, Aniê Ieda Rodriguez, Maria Constanza |
dc.subject.por.fl_str_mv |
PCR em tempo real Sílica Chelex-100 Salmonella. PCR en tiempo real Sílica Chelex-100 Salmonella. Real-time PCR Silica particles Chelex-100 resin Salmonella. |
topic |
PCR em tempo real Sílica Chelex-100 Salmonella. PCR en tiempo real Sílica Chelex-100 Salmonella. Real-time PCR Silica particles Chelex-100 resin Salmonella. |
description |
There are many extraction methods available to purify bacterial DNA. PCR efficiency can vary depending on the quantity and quality of the template DNA used. This study evaluated the efficiency, quality, cost and rapidness of two in-house protocols, silica particles and Chelex-100 resin, for the extraction of twenty Salmonella isolates from boot swabs. The aim of this experiment was to compare the extraction methods for Salmonella spp. detection. DNA extraction was performed for each method and subjected to real-time PCR amplification. The amounts and integrity of DNA were determined using spectrophotometry and agarose gel electrophoresis, and the efficiency measured with real-time PCR. Limit of Detection (LOD) was determined with serial dilutions of a S. Typhimurium reference strain resulting in linear regression coefficients of R 2=0.992 (efficiency 119.31) for silica and R 2=0.958 (efficiency 93.33) for Chelex, with LOD of 10 -4 for both. Silica particles method resulted in higher DNA yield, 85.01 ± 59.11 compared to 50.74 ± 12.95 and 260/280 ratio, 1.77 ± 0.02 versus 1.63 ± 0.13. DNA integrity was superior using silica, gel showed a unique band higher than 2.000 bp, while Chelex-100 was imperceptive or degraded. Regarding PCR results, the mean quantification cycle (Cq) for silica was 17.08 ± 0.73 and Chelex-100, 17.64 ±0.56 (suggesting lower DNA values). Results showed that both methods were effective for the DNA extraction of Salmonella, once PCR resulted positive for all samples, efficiency was higher for silica. Chelex-100 resin was performed in less time at a lower cost. |
publishDate |
2023 |
dc.date.none.fl_str_mv |
2023-01-12 |
dc.type.driver.fl_str_mv |
info:eu-repo/semantics/article info:eu-repo/semantics/publishedVersion |
format |
article |
status_str |
publishedVersion |
dc.identifier.uri.fl_str_mv |
https://rsdjournal.org/index.php/rsd/article/view/39618 10.33448/rsd-v12i1.39618 |
url |
https://rsdjournal.org/index.php/rsd/article/view/39618 |
identifier_str_mv |
10.33448/rsd-v12i1.39618 |
dc.language.iso.fl_str_mv |
eng |
language |
eng |
dc.relation.none.fl_str_mv |
https://rsdjournal.org/index.php/rsd/article/view/39618/32620 |
dc.rights.driver.fl_str_mv |
https://creativecommons.org/licenses/by/4.0 info:eu-repo/semantics/openAccess |
rights_invalid_str_mv |
https://creativecommons.org/licenses/by/4.0 |
eu_rights_str_mv |
openAccess |
dc.format.none.fl_str_mv |
application/pdf |
dc.publisher.none.fl_str_mv |
Research, Society and Development |
publisher.none.fl_str_mv |
Research, Society and Development |
dc.source.none.fl_str_mv |
Research, Society and Development; Vol. 12 No. 1; e28312139618 Research, Society and Development; Vol. 12 Núm. 1; e28312139618 Research, Society and Development; v. 12 n. 1; e28312139618 2525-3409 reponame:Research, Society and Development instname:Universidade Federal de Itajubá (UNIFEI) instacron:UNIFEI |
instname_str |
Universidade Federal de Itajubá (UNIFEI) |
instacron_str |
UNIFEI |
institution |
UNIFEI |
reponame_str |
Research, Society and Development |
collection |
Research, Society and Development |
repository.name.fl_str_mv |
Research, Society and Development - Universidade Federal de Itajubá (UNIFEI) |
repository.mail.fl_str_mv |
rsd.articles@gmail.com |
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1797052616041037824 |