EVALUATION OF FOUR DIFFERENT DNA EXTRACTION METHODS IN COAGULASE-NEGATIVE STAPHYLOCOCCI CLINICAL ISOLATES
Autor(a) principal: | |
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Data de Publicação: | 2014 |
Outros Autores: | , , , |
Tipo de documento: | Artigo |
Idioma: | eng |
Título da fonte: | Revista do Instituto de Medicina Tropical de São Paulo |
Texto Completo: | https://www.revistas.usp.br/rimtsp/article/view/78693 |
Resumo: | Currently there are several methods to extract bacterial DNA based on different principles. However, the amount and the quality of the DNA obtained by each one of those methods is highly variable and microorganism dependent, as illustrated by coagulase-negative staphylococci (CoNS) which have a thick cell wall that is difficult to lyse. This study was designed to compare the quality and the amount of CoNS DNA, extracted by four different techniques: two in-house protocols and two commercial kits. DNA amount and quality determination was performed through spectrophotometry. The extracted DNA was also analyzed using agarose gel electrophoresis and by PCR. 267 isolates of CoNS were used in this study. The column method and thermal lyses showed better results with regard to DNA quality (mean ratio of A260/280 = 1.95) and average concentration of DNA (), respectively. All four methods tested provided appropriate DNA for PCR amplification, but with different yields. DNA quality is important since it allows the application of a large number of molecular biology techniques, and also it's storage for a longer period of time. In this sense the extraction method based on an extraction column presented the best results for CoNS. |
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EVALUATION OF FOUR DIFFERENT DNA EXTRACTION METHODS IN COAGULASE-NEGATIVE STAPHYLOCOCCI CLINICAL ISOLATES Avaliacao de quatro metodos diferentes de extracao de DNA em isolados clinicos de estafilococos coagulase negativos (SCoN) Currently there are several methods to extract bacterial DNA based on different principles. However, the amount and the quality of the DNA obtained by each one of those methods is highly variable and microorganism dependent, as illustrated by coagulase-negative staphylococci (CoNS) which have a thick cell wall that is difficult to lyse. This study was designed to compare the quality and the amount of CoNS DNA, extracted by four different techniques: two in-house protocols and two commercial kits. DNA amount and quality determination was performed through spectrophotometry. The extracted DNA was also analyzed using agarose gel electrophoresis and by PCR. 267 isolates of CoNS were used in this study. The column method and thermal lyses showed better results with regard to DNA quality (mean ratio of A260/280 = 1.95) and average concentration of DNA (), respectively. All four methods tested provided appropriate DNA for PCR amplification, but with different yields. DNA quality is important since it allows the application of a large number of molecular biology techniques, and also it's storage for a longer period of time. In this sense the extraction method based on an extraction column presented the best results for CoNS. Atualmente, para extrair o DNA bacteriano, existem diversos métodos baseados em diferentes princípios. Entretanto, a quantidade e qualidade do DNA obtido por cada um destes métodos é variável e depende do tipo de micro-organismo em questão; os estafilococos coagulase-negativos (CoNS), por exemplo, possuem parede celular espessa difícil de lisar. O objetivo deste estudo foi comparar a quantidade e a qualidade do DNA extraído de isolados clínicos de CoNS utilizando quatro metodologias diferentes: dois protocolos caseiros e dois kits comerciais. A determinação da quantidade e da qualidade do DNA foi realizada por espectrofotometria. O DNA extraído também foi analisado em eletroforese em gel de agarose e por PCR. A concentração média de DNA foi mais alta no método de lise térmica (). Entretanto, com relação à qualidade do DNA, o kit comercial que utiliza um método de extração baseado em uma coluna de separação apresentou melhor resultado (média da relação A260/280 = 1,95). As quatro técnicas testadas forneceram DNA passível de amplificação por PCR, porém com diferentes rendimentos. A qualidade do DNA extraído de bactérias é importante, pois possibilita a realização de maior número de técnicas de biologia molecular e também armazenamento do material por maior período de tempo. Neste sentido, a técnica de extração por coluna de separação apresentou melhor desempenho frente aos CoNS. Universidade de São Paulo. Instituto de Medicina Tropical de São Paulo2014-01-01info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionapplication/pdfhttps://www.revistas.usp.br/rimtsp/article/view/78693Revista do Instituto de Medicina Tropical de São Paulo; Vol. 56 No. 1 (2014); 29-33Revista do Instituto de Medicina Tropical de São Paulo; Vol. 56 Núm. 1 (2014); 29-33Revista do Instituto de Medicina Tropical de São Paulo; v. 56 n. 1 (2014); 29-331678-99460036-4665reponame:Revista do Instituto de Medicina Tropical de São Pauloinstname:Instituto de Medicina Tropical (IMT)instacron:IMTenghttps://www.revistas.usp.br/rimtsp/article/view/78693/82745Copyright (c) 2018 Revista do Instituto de Medicina Tropical de São Pauloinfo:eu-repo/semantics/openAccessOliveira, Caio Fernando de Paim, Thiago Galvao da Silva Reiter, Keli Cristine Rieger, Alexandre D'azevedo, Pedro Alves 2014-04-03T19:06:41Zoai:revistas.usp.br:article/78693Revistahttp://www.revistas.usp.br/rimtsp/indexPUBhttps://www.revistas.usp.br/rimtsp/oai||revimtsp@usp.br1678-99460036-4665opendoar:2022-12-13T16:52:15.949184Revista do Instituto de Medicina Tropical de São Paulo - Instituto de Medicina Tropical (IMT)true |
dc.title.none.fl_str_mv |
EVALUATION OF FOUR DIFFERENT DNA EXTRACTION METHODS IN COAGULASE-NEGATIVE STAPHYLOCOCCI CLINICAL ISOLATES Avaliacao de quatro metodos diferentes de extracao de DNA em isolados clinicos de estafilococos coagulase negativos (SCoN) |
title |
EVALUATION OF FOUR DIFFERENT DNA EXTRACTION METHODS IN COAGULASE-NEGATIVE STAPHYLOCOCCI CLINICAL ISOLATES |
spellingShingle |
EVALUATION OF FOUR DIFFERENT DNA EXTRACTION METHODS IN COAGULASE-NEGATIVE STAPHYLOCOCCI CLINICAL ISOLATES Oliveira, Caio Fernando de |
title_short |
EVALUATION OF FOUR DIFFERENT DNA EXTRACTION METHODS IN COAGULASE-NEGATIVE STAPHYLOCOCCI CLINICAL ISOLATES |
title_full |
EVALUATION OF FOUR DIFFERENT DNA EXTRACTION METHODS IN COAGULASE-NEGATIVE STAPHYLOCOCCI CLINICAL ISOLATES |
title_fullStr |
EVALUATION OF FOUR DIFFERENT DNA EXTRACTION METHODS IN COAGULASE-NEGATIVE STAPHYLOCOCCI CLINICAL ISOLATES |
title_full_unstemmed |
EVALUATION OF FOUR DIFFERENT DNA EXTRACTION METHODS IN COAGULASE-NEGATIVE STAPHYLOCOCCI CLINICAL ISOLATES |
title_sort |
EVALUATION OF FOUR DIFFERENT DNA EXTRACTION METHODS IN COAGULASE-NEGATIVE STAPHYLOCOCCI CLINICAL ISOLATES |
author |
Oliveira, Caio Fernando de |
author_facet |
Oliveira, Caio Fernando de Paim, Thiago Galvao da Silva Reiter, Keli Cristine Rieger, Alexandre D'azevedo, Pedro Alves |
author_role |
author |
author2 |
Paim, Thiago Galvao da Silva Reiter, Keli Cristine Rieger, Alexandre D'azevedo, Pedro Alves |
author2_role |
author author author author |
dc.contributor.author.fl_str_mv |
Oliveira, Caio Fernando de Paim, Thiago Galvao da Silva Reiter, Keli Cristine Rieger, Alexandre D'azevedo, Pedro Alves |
description |
Currently there are several methods to extract bacterial DNA based on different principles. However, the amount and the quality of the DNA obtained by each one of those methods is highly variable and microorganism dependent, as illustrated by coagulase-negative staphylococci (CoNS) which have a thick cell wall that is difficult to lyse. This study was designed to compare the quality and the amount of CoNS DNA, extracted by four different techniques: two in-house protocols and two commercial kits. DNA amount and quality determination was performed through spectrophotometry. The extracted DNA was also analyzed using agarose gel electrophoresis and by PCR. 267 isolates of CoNS were used in this study. The column method and thermal lyses showed better results with regard to DNA quality (mean ratio of A260/280 = 1.95) and average concentration of DNA (), respectively. All four methods tested provided appropriate DNA for PCR amplification, but with different yields. DNA quality is important since it allows the application of a large number of molecular biology techniques, and also it's storage for a longer period of time. In this sense the extraction method based on an extraction column presented the best results for CoNS. |
publishDate |
2014 |
dc.date.none.fl_str_mv |
2014-01-01 |
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://www.revistas.usp.br/rimtsp/article/view/78693 |
url |
https://www.revistas.usp.br/rimtsp/article/view/78693 |
dc.language.iso.fl_str_mv |
eng |
language |
eng |
dc.relation.none.fl_str_mv |
https://www.revistas.usp.br/rimtsp/article/view/78693/82745 |
dc.rights.driver.fl_str_mv |
Copyright (c) 2018 Revista do Instituto de Medicina Tropical de São Paulo info:eu-repo/semantics/openAccess |
rights_invalid_str_mv |
Copyright (c) 2018 Revista do Instituto de Medicina Tropical de São Paulo |
eu_rights_str_mv |
openAccess |
dc.format.none.fl_str_mv |
application/pdf |
dc.publisher.none.fl_str_mv |
Universidade de São Paulo. Instituto de Medicina Tropical de São Paulo |
publisher.none.fl_str_mv |
Universidade de São Paulo. Instituto de Medicina Tropical de São Paulo |
dc.source.none.fl_str_mv |
Revista do Instituto de Medicina Tropical de São Paulo; Vol. 56 No. 1 (2014); 29-33 Revista do Instituto de Medicina Tropical de São Paulo; Vol. 56 Núm. 1 (2014); 29-33 Revista do Instituto de Medicina Tropical de São Paulo; v. 56 n. 1 (2014); 29-33 1678-9946 0036-4665 reponame:Revista do Instituto de Medicina Tropical de São Paulo instname:Instituto de Medicina Tropical (IMT) instacron:IMT |
instname_str |
Instituto de Medicina Tropical (IMT) |
instacron_str |
IMT |
institution |
IMT |
reponame_str |
Revista do Instituto de Medicina Tropical de São Paulo |
collection |
Revista do Instituto de Medicina Tropical de São Paulo |
repository.name.fl_str_mv |
Revista do Instituto de Medicina Tropical de São Paulo - Instituto de Medicina Tropical (IMT) |
repository.mail.fl_str_mv |
||revimtsp@usp.br |
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1798951649026244608 |