Effects of tissue handling and processing steps on PCR for detection of Mycobacterium tuberculosis in formalin-fixed paraffin-embedded samples
Autor(a) principal: | |
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Data de Publicação: | 2008 |
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/31213 |
Resumo: | Development and standardization of reliable methods for detection of Mycobacterium tuberculosis in clinical samples is an important goal in laboratories throughout the world. In this work, lung and spleen fragments from a patient who died with the diagnosis of miliary tuberculosis were used to evaluate the influence of the type of fixative as well as the fixation and paraffin inclusion protocols on PCR performance in paraffin embedded specimens. Tissue fragments were fixed for four h to 48 h, using either 10% non-buffered or 10% buffered formalin, and embedded in pure paraffin or paraffin mixed with bee wax. Specimens were submitted to PCR for amplification of the human beta-actin gene and separately for amplification of the insertion sequence IS6110, specific from the M. tuberculosis complex. Amplification of the beta-actin gene was positive in all samples. No amplicons were generated by PCR-IS6110 when lung tissue fragments were fixed using 10% non-buffered formalin and were embedded in paraffin containing bee wax. In conclusion, combined inhibitory factors interfere in the detection of M. tuberculosis in stored material. It is important to control these inhibitory factors in order to implement molecular diagnosis in pathology laboratories. |
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Effects of tissue handling and processing steps on PCR for detection of Mycobacterium tuberculosis in formalin-fixed paraffin-embedded samples Efeitos das etapas de tratamento e processamento do tecido na PCR para detecção de Mycobacterium tuberculosis em amostras fixadas em formalina e incluídas em parafina TuberculosisPCRDiagnosisParaffin-embedded tissue Development and standardization of reliable methods for detection of Mycobacterium tuberculosis in clinical samples is an important goal in laboratories throughout the world. In this work, lung and spleen fragments from a patient who died with the diagnosis of miliary tuberculosis were used to evaluate the influence of the type of fixative as well as the fixation and paraffin inclusion protocols on PCR performance in paraffin embedded specimens. Tissue fragments were fixed for four h to 48 h, using either 10% non-buffered or 10% buffered formalin, and embedded in pure paraffin or paraffin mixed with bee wax. Specimens were submitted to PCR for amplification of the human beta-actin gene and separately for amplification of the insertion sequence IS6110, specific from the M. tuberculosis complex. Amplification of the beta-actin gene was positive in all samples. No amplicons were generated by PCR-IS6110 when lung tissue fragments were fixed using 10% non-buffered formalin and were embedded in paraffin containing bee wax. In conclusion, combined inhibitory factors interfere in the detection of M. tuberculosis in stored material. It is important to control these inhibitory factors in order to implement molecular diagnosis in pathology laboratories. O desenvolvimento e a padronização de métodos confiáveis para a detecção de Mycobacterium tuberculosis em amostras clínicas é um objetivo importante nos laboratórios de todo o mundo. Neste trabalho, fragmentos de pulmão e baço de paciente que morreu com o diagnóstico de tuberculose miliar foram usados para avaliar a influência do tipo de fixador e dos protocolos de fixação e inclusão em parafina na performance da PCR. Fragmentos de tecido foram fixados por quatro h a 48 h, usando formalina não tamponada a 10% ou formalina tamponada a 10% e incluídos em parafina pura ou misturada a cera de abelha. As amostras foram submetidas a PCR para amplificação do gene da beta-actina humana e, separadamente, para amplificação da sequência de inserção IS6110, específica do complexo M. tuberculosis. O resultado da amplificação do gene da beta-actina foi positivo em todas as amostras. Não foram gerados amplicons na PCR-IS6110 em amostras de tecido pulmonar fixadas usando formalina não tamponada a 10% e incluídas em parafina com cera de abelha. Em conclusão, fatores inibitórios combinados interferiram na detecção de M. tuberculosis em material de arquivo. É importante controlar estes fatores inibitórios para poder implementar o diagnóstico molecular em laboratórios de patologia. Universidade de São Paulo. Instituto de Medicina Tropical de São Paulo2008-12-01info:eu-repo/semantics/articleinfo:eu-repo/semantics/publishedVersionapplication/pdfhttps://www.revistas.usp.br/rimtsp/article/view/31213Revista do Instituto de Medicina Tropical de São Paulo; Vol. 50 No. 6 (2008); 321-326 Revista do Instituto de Medicina Tropical de São Paulo; Vol. 50 Núm. 6 (2008); 321-326 Revista do Instituto de Medicina Tropical de São Paulo; v. 50 n. 6 (2008); 321-326 1678-99460036-4665reponame:Revista do Instituto de Medicina Tropical de São Pauloinstname:Instituto de Medicina Tropical (IMT)instacron:IMTenghttps://www.revistas.usp.br/rimtsp/article/view/31213/33097Copyright (c) 2018 Revista do Instituto de Medicina Tropical de São Pauloinfo:eu-repo/semantics/openAccessBarcelos, DeniseFranco, Marcello F.Leão, Sylvia Cardoso2012-07-07T19:12:26Zoai:revistas.usp.br:article/31213Revistahttp://www.revistas.usp.br/rimtsp/indexPUBhttps://www.revistas.usp.br/rimtsp/oai||revimtsp@usp.br1678-99460036-4665opendoar:2022-12-13T16:51:53.871313Revista do Instituto de Medicina Tropical de São Paulo - Instituto de Medicina Tropical (IMT)true |
dc.title.none.fl_str_mv |
Effects of tissue handling and processing steps on PCR for detection of Mycobacterium tuberculosis in formalin-fixed paraffin-embedded samples Efeitos das etapas de tratamento e processamento do tecido na PCR para detecção de Mycobacterium tuberculosis em amostras fixadas em formalina e incluídas em parafina |
title |
Effects of tissue handling and processing steps on PCR for detection of Mycobacterium tuberculosis in formalin-fixed paraffin-embedded samples |
spellingShingle |
Effects of tissue handling and processing steps on PCR for detection of Mycobacterium tuberculosis in formalin-fixed paraffin-embedded samples Barcelos, Denise Tuberculosis PCR Diagnosis Paraffin-embedded tissue |
title_short |
Effects of tissue handling and processing steps on PCR for detection of Mycobacterium tuberculosis in formalin-fixed paraffin-embedded samples |
title_full |
Effects of tissue handling and processing steps on PCR for detection of Mycobacterium tuberculosis in formalin-fixed paraffin-embedded samples |
title_fullStr |
Effects of tissue handling and processing steps on PCR for detection of Mycobacterium tuberculosis in formalin-fixed paraffin-embedded samples |
title_full_unstemmed |
Effects of tissue handling and processing steps on PCR for detection of Mycobacterium tuberculosis in formalin-fixed paraffin-embedded samples |
title_sort |
Effects of tissue handling and processing steps on PCR for detection of Mycobacterium tuberculosis in formalin-fixed paraffin-embedded samples |
author |
Barcelos, Denise |
author_facet |
Barcelos, Denise Franco, Marcello F. Leão, Sylvia Cardoso |
author_role |
author |
author2 |
Franco, Marcello F. Leão, Sylvia Cardoso |
author2_role |
author author |
dc.contributor.author.fl_str_mv |
Barcelos, Denise Franco, Marcello F. Leão, Sylvia Cardoso |
dc.subject.por.fl_str_mv |
Tuberculosis PCR Diagnosis Paraffin-embedded tissue |
topic |
Tuberculosis PCR Diagnosis Paraffin-embedded tissue |
description |
Development and standardization of reliable methods for detection of Mycobacterium tuberculosis in clinical samples is an important goal in laboratories throughout the world. In this work, lung and spleen fragments from a patient who died with the diagnosis of miliary tuberculosis were used to evaluate the influence of the type of fixative as well as the fixation and paraffin inclusion protocols on PCR performance in paraffin embedded specimens. Tissue fragments were fixed for four h to 48 h, using either 10% non-buffered or 10% buffered formalin, and embedded in pure paraffin or paraffin mixed with bee wax. Specimens were submitted to PCR for amplification of the human beta-actin gene and separately for amplification of the insertion sequence IS6110, specific from the M. tuberculosis complex. Amplification of the beta-actin gene was positive in all samples. No amplicons were generated by PCR-IS6110 when lung tissue fragments were fixed using 10% non-buffered formalin and were embedded in paraffin containing bee wax. In conclusion, combined inhibitory factors interfere in the detection of M. tuberculosis in stored material. It is important to control these inhibitory factors in order to implement molecular diagnosis in pathology laboratories. |
publishDate |
2008 |
dc.date.none.fl_str_mv |
2008-12-01 |
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/31213 |
url |
https://www.revistas.usp.br/rimtsp/article/view/31213 |
dc.language.iso.fl_str_mv |
eng |
language |
eng |
dc.relation.none.fl_str_mv |
https://www.revistas.usp.br/rimtsp/article/view/31213/33097 |
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. 50 No. 6 (2008); 321-326 Revista do Instituto de Medicina Tropical de São Paulo; Vol. 50 Núm. 6 (2008); 321-326 Revista do Instituto de Medicina Tropical de São Paulo; v. 50 n. 6 (2008); 321-326 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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