A comparison of three DNA extractive procedures with Leptospira for polymerase chain reaction analysis

Detalhes bibliográficos
Autor(a) principal: Veloso,IF
Data de Publicação: 2000
Outros Autores: Lopes,MTP, Salas,CE, Moreira,EC
Tipo de documento: Artigo
Idioma: eng
Título da fonte: Memórias do Instituto Oswaldo Cruz
Texto Completo: http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0074-02762000000300008
Resumo: Three DNA extraction methods were evaluated in this study: proteinase K followed by phenol-chloroform; a plant proteinase (E6870) followed by phenol-chloroform; and boiling of leptospires in 0.1 mM Tris, pH 7.0 for 10 min at 100°C, with no phenol treatment. Every strain treated with proteinase K or E6870 afforded positive polymerase chain reaction (PCR) reaction. On the other hand, from five strains extracted by the boiling method, three did not feature the 849 bp band characteristic in Leptospira. We also evaluated by RAPD-PCR, DNAs from serovars isolated with proteinase K and proteinase 6870 with primers B11/B12. Each of the DNA samples provided PCR profiles in agreement with previous data. Moreover, the results with E6870 showed less background non-specific amplification, suggesting that removal of nucleases was more efficient with E6870. The limit for detection by PCR using Lep13/Lep14 was determined to be 10(2) leptospira, using the silver stain procedure.
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spelling A comparison of three DNA extractive procedures with Leptospira for polymerase chain reaction analysispolymerase chain reactionleptospiraDNA extractionThree DNA extraction methods were evaluated in this study: proteinase K followed by phenol-chloroform; a plant proteinase (E6870) followed by phenol-chloroform; and boiling of leptospires in 0.1 mM Tris, pH 7.0 for 10 min at 100°C, with no phenol treatment. Every strain treated with proteinase K or E6870 afforded positive polymerase chain reaction (PCR) reaction. On the other hand, from five strains extracted by the boiling method, three did not feature the 849 bp band characteristic in Leptospira. We also evaluated by RAPD-PCR, DNAs from serovars isolated with proteinase K and proteinase 6870 with primers B11/B12. Each of the DNA samples provided PCR profiles in agreement with previous data. Moreover, the results with E6870 showed less background non-specific amplification, suggesting that removal of nucleases was more efficient with E6870. The limit for detection by PCR using Lep13/Lep14 was determined to be 10(2) leptospira, using the silver stain procedure.Instituto Oswaldo Cruz, Ministério da Saúde2000-06-01info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersiontext/htmlhttp://www.scielo.br/scielo.php?script=sci_arttext&pid=S0074-02762000000300008Memórias do Instituto Oswaldo Cruz v.95 n.3 2000reponame:Memórias do Instituto Oswaldo Cruzinstname:Fundação Oswaldo Cruzinstacron:FIOCRUZ10.1590/S0074-02762000000300008info:eu-repo/semantics/openAccessVeloso,IFLopes,MTPSalas,CEMoreira,ECeng2020-04-25T17:48:23Zhttp://www.scielo.br/oai/scielo-oai.php0074-02761678-8060opendoar:null2020-04-26 02:09:39.576Memórias do Instituto Oswaldo Cruz - Fundação Oswaldo Cruztrue
dc.title.none.fl_str_mv A comparison of three DNA extractive procedures with Leptospira for polymerase chain reaction analysis
title A comparison of three DNA extractive procedures with Leptospira for polymerase chain reaction analysis
spellingShingle A comparison of three DNA extractive procedures with Leptospira for polymerase chain reaction analysis
Veloso,IF
polymerase chain reaction
leptospira
DNA extraction
title_short A comparison of three DNA extractive procedures with Leptospira for polymerase chain reaction analysis
title_full A comparison of three DNA extractive procedures with Leptospira for polymerase chain reaction analysis
title_fullStr A comparison of three DNA extractive procedures with Leptospira for polymerase chain reaction analysis
title_full_unstemmed A comparison of three DNA extractive procedures with Leptospira for polymerase chain reaction analysis
title_sort A comparison of three DNA extractive procedures with Leptospira for polymerase chain reaction analysis
author Veloso,IF
author_facet Veloso,IF
Lopes,MTP
Salas,CE
Moreira,EC
author_role author
author2 Lopes,MTP
Salas,CE
Moreira,EC
author2_role author
author
author
dc.contributor.author.fl_str_mv Veloso,IF
Lopes,MTP
Salas,CE
Moreira,EC
dc.subject.por.fl_str_mv polymerase chain reaction
leptospira
DNA extraction
topic polymerase chain reaction
leptospira
DNA extraction
dc.description.none.fl_txt_mv Three DNA extraction methods were evaluated in this study: proteinase K followed by phenol-chloroform; a plant proteinase (E6870) followed by phenol-chloroform; and boiling of leptospires in 0.1 mM Tris, pH 7.0 for 10 min at 100°C, with no phenol treatment. Every strain treated with proteinase K or E6870 afforded positive polymerase chain reaction (PCR) reaction. On the other hand, from five strains extracted by the boiling method, three did not feature the 849 bp band characteristic in Leptospira. We also evaluated by RAPD-PCR, DNAs from serovars isolated with proteinase K and proteinase 6870 with primers B11/B12. Each of the DNA samples provided PCR profiles in agreement with previous data. Moreover, the results with E6870 showed less background non-specific amplification, suggesting that removal of nucleases was more efficient with E6870. The limit for detection by PCR using Lep13/Lep14 was determined to be 10(2) leptospira, using the silver stain procedure.
description Three DNA extraction methods were evaluated in this study: proteinase K followed by phenol-chloroform; a plant proteinase (E6870) followed by phenol-chloroform; and boiling of leptospires in 0.1 mM Tris, pH 7.0 for 10 min at 100°C, with no phenol treatment. Every strain treated with proteinase K or E6870 afforded positive polymerase chain reaction (PCR) reaction. On the other hand, from five strains extracted by the boiling method, three did not feature the 849 bp band characteristic in Leptospira. We also evaluated by RAPD-PCR, DNAs from serovars isolated with proteinase K and proteinase 6870 with primers B11/B12. Each of the DNA samples provided PCR profiles in agreement with previous data. Moreover, the results with E6870 showed less background non-specific amplification, suggesting that removal of nucleases was more efficient with E6870. The limit for detection by PCR using Lep13/Lep14 was determined to be 10(2) leptospira, using the silver stain procedure.
publishDate 2000
dc.date.none.fl_str_mv 2000-06-01
dc.type.driver.fl_str_mv info:eu-repo/semantics/article
dc.type.status.fl_str_mv info:eu-repo/semantics/publishedVersion
format article
status_str publishedVersion
dc.identifier.uri.fl_str_mv http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0074-02762000000300008
url http://www.scielo.br/scielo.php?script=sci_arttext&pid=S0074-02762000000300008
dc.language.iso.fl_str_mv eng
language eng
dc.relation.none.fl_str_mv 10.1590/S0074-02762000000300008
dc.rights.driver.fl_str_mv info:eu-repo/semantics/openAccess
eu_rights_str_mv openAccess
dc.format.none.fl_str_mv text/html
dc.publisher.none.fl_str_mv Instituto Oswaldo Cruz, Ministério da Saúde
publisher.none.fl_str_mv Instituto Oswaldo Cruz, Ministério da Saúde
dc.source.none.fl_str_mv Memórias do Instituto Oswaldo Cruz v.95 n.3 2000
reponame:Memórias do Instituto Oswaldo Cruz
instname:Fundação Oswaldo Cruz
instacron:FIOCRUZ
reponame_str Memórias do Instituto Oswaldo Cruz
collection Memórias do Instituto Oswaldo Cruz
instname_str Fundação Oswaldo Cruz
instacron_str FIOCRUZ
institution FIOCRUZ
repository.name.fl_str_mv Memórias do Instituto Oswaldo Cruz - Fundação Oswaldo Cruz
repository.mail.fl_str_mv
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