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Processing of long-stored archival cervical smears for human papillomavirus detection by the polymerase chain reaction.
The efficiency of a freeze-thaw method, a proteinase K/Tween 20 lysis method and a guanidinium isothiocyanate/silica beads method for DNA extraction from fixed and Papanicolaou-stained cells from the cervical cancer cell line Siha was measured by beta-globin polymerase chain reaction (PCR). The GTC/...
Autores principales: | , , , , , |
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Formato: | Texto |
Lenguaje: | English |
Publicado: |
Nature Publishing Group
1995
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2033993/ https://www.ncbi.nlm.nih.gov/pubmed/7543772 |
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author | de Roda Husman, A. M. Snijders, P. J. Stel, H. V. van den Brule, A. J. Meijer, C. J. Walboomers, J. M. |
author_facet | de Roda Husman, A. M. Snijders, P. J. Stel, H. V. van den Brule, A. J. Meijer, C. J. Walboomers, J. M. |
author_sort | de Roda Husman, A. M. |
collection | PubMed |
description | The efficiency of a freeze-thaw method, a proteinase K/Tween 20 lysis method and a guanidinium isothiocyanate/silica beads method for DNA extraction from fixed and Papanicolaou-stained cells from the cervical cancer cell line Siha was measured by beta-globin polymerase chain reaction (PCR). The GTC/silica beads method, which appeared superior, revealed a human papillomavirus (HPV) general primer-mediated PCR sensitivity of 50-500 copies of HPV 16 per sample using dilutions of fixed and stained Siha cells. Application to archival cervical smears (n = 116) revealed that the yield and size of amplifiable DNA decreases with storage time. The longer the storage time, the more repetitions of the whole procedure, including the lysis step, were required to extract sufficient amplifiable DNA. In this way, an overall beta-globin PCR positivity for 98% of the smears was reached. Further analysis revealed that a maximum size of 200 bp could be amplified from smears stored for up to 9 years. The method was validated by demonstrating by PCR the same HPV types in archival smears and corresponding cervical biopsies of cervical cancer patients. In conclusion, the GTC/silica beads method appears suitable to process archival cervical smears for HPV detection by PCR. provided that stepwise adjustments are made until beta-globin PCR positivity is obtained and primers are chosen which amplify a maximum of about 200 bp. IMAGES: |
format | Text |
id | pubmed-2033993 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 1995 |
publisher | Nature Publishing Group |
record_format | MEDLINE/PubMed |
spelling | pubmed-20339932009-09-10 Processing of long-stored archival cervical smears for human papillomavirus detection by the polymerase chain reaction. de Roda Husman, A. M. Snijders, P. J. Stel, H. V. van den Brule, A. J. Meijer, C. J. Walboomers, J. M. Br J Cancer Research Article The efficiency of a freeze-thaw method, a proteinase K/Tween 20 lysis method and a guanidinium isothiocyanate/silica beads method for DNA extraction from fixed and Papanicolaou-stained cells from the cervical cancer cell line Siha was measured by beta-globin polymerase chain reaction (PCR). The GTC/silica beads method, which appeared superior, revealed a human papillomavirus (HPV) general primer-mediated PCR sensitivity of 50-500 copies of HPV 16 per sample using dilutions of fixed and stained Siha cells. Application to archival cervical smears (n = 116) revealed that the yield and size of amplifiable DNA decreases with storage time. The longer the storage time, the more repetitions of the whole procedure, including the lysis step, were required to extract sufficient amplifiable DNA. In this way, an overall beta-globin PCR positivity for 98% of the smears was reached. Further analysis revealed that a maximum size of 200 bp could be amplified from smears stored for up to 9 years. The method was validated by demonstrating by PCR the same HPV types in archival smears and corresponding cervical biopsies of cervical cancer patients. In conclusion, the GTC/silica beads method appears suitable to process archival cervical smears for HPV detection by PCR. provided that stepwise adjustments are made until beta-globin PCR positivity is obtained and primers are chosen which amplify a maximum of about 200 bp. IMAGES: Nature Publishing Group 1995-08 /pmc/articles/PMC2033993/ /pubmed/7543772 Text en https://creativecommons.org/licenses/by/4.0/This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit https://creativecommons.org/licenses/by/4.0/. |
spellingShingle | Research Article de Roda Husman, A. M. Snijders, P. J. Stel, H. V. van den Brule, A. J. Meijer, C. J. Walboomers, J. M. Processing of long-stored archival cervical smears for human papillomavirus detection by the polymerase chain reaction. |
title | Processing of long-stored archival cervical smears for human papillomavirus detection by the polymerase chain reaction. |
title_full | Processing of long-stored archival cervical smears for human papillomavirus detection by the polymerase chain reaction. |
title_fullStr | Processing of long-stored archival cervical smears for human papillomavirus detection by the polymerase chain reaction. |
title_full_unstemmed | Processing of long-stored archival cervical smears for human papillomavirus detection by the polymerase chain reaction. |
title_short | Processing of long-stored archival cervical smears for human papillomavirus detection by the polymerase chain reaction. |
title_sort | processing of long-stored archival cervical smears for human papillomavirus detection by the polymerase chain reaction. |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2033993/ https://www.ncbi.nlm.nih.gov/pubmed/7543772 |
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