A comparison of three DNA extractive procedures with Leptospira for polymerase chain reaction analysis
Autor(a) principal: | |
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Data de Publicação: | 2000 |
Outros Autores: | , , |
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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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 |
|
_version_ |
1669937679679094784 |