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Monkeypox virus detection in rodents using real-time 3′-minor groove binder TaqMan® assays on the Roche LightCycler
During the summer of 2003, an outbreak of human monkeypox occurred in the Midwest region of the United States. In all, 52 rodents suspected of being infected with monkeypox virus were collected from an exotic pet dealer and from private homes. The rodents were euthanized and submitted for testing to...
Autores principales: | , , , , , , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
United States & Canadian Academy of Pathology.
2004
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9827366/ https://www.ncbi.nlm.nih.gov/pubmed/15208646 http://dx.doi.org/10.1038/labinvest.3700143 |
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author | Kulesh, David A Loveless, Bonnie M Norwood, David Garrison, Jeffrey Whitehouse, Chris A Hartmann, Chris Mucker, Eric Miller, David Wasieloski, Leonard P Huggins, John Huhn, Gregory Miser, Lori L Imig, Carroll Martinez, Mark Larsen, Tom Rossi, Cynthia A Ludwig, George V |
author_facet | Kulesh, David A Loveless, Bonnie M Norwood, David Garrison, Jeffrey Whitehouse, Chris A Hartmann, Chris Mucker, Eric Miller, David Wasieloski, Leonard P Huggins, John Huhn, Gregory Miser, Lori L Imig, Carroll Martinez, Mark Larsen, Tom Rossi, Cynthia A Ludwig, George V |
author_sort | Kulesh, David A |
collection | PubMed |
description | During the summer of 2003, an outbreak of human monkeypox occurred in the Midwest region of the United States. In all, 52 rodents suspected of being infected with monkeypox virus were collected from an exotic pet dealer and from private homes. The rodents were euthanized and submitted for testing to the United States Army Medical Research Institute of Infectious Diseases by the Galesburg Animal Disease Laboratory, Illinois Department of Agriculture. The rodent tissue samples were appropriately processed and then tested by using an integrated approach involving real-time polymerase chain reaction (PCR) assays, an antigen-detection immunoassay, and virus culture. We designed and extensively tested two specific real-time PCR assays for rapidly detecting monkeypox virus DNA using the Vaccinia virus F3L and N3R genes as targets. The assays were validated against panels of orthopox viral and miscellaneous bacterial DNAs. A pan-orthopox electrochemiluminescence (ECL) assay was used to further confirm the presence of Orthopoxvirus infection of the rodents. Seven of 12 (58%) animals (seven of 52 (15%) of all animals) tested positive in both monkeypox-specific PCR assays and two additional pan-orthopox PCR assays (in at least one tissue). The ECL results showed varying degrees of agreement with PCR. One hamster and three gerbils were positive by both PCR and ECL for all tissues tested. In addition, we attempted to verify the presence of monkeypox virus by culture on multiple cell lines, by immunohistology, and by electron microscopy, with negative results. Sequencing the PCR products from the samples indicated 100% identity with monkeypox virus strain Zaire-96-I-16 (a human isolate from the Congo). These real-time PCR and ECL assays represent a significant addition to the battery of tests for the detection of various orthopoxviruses. In light of the recent monkeypox virus transmissions, early detection of the virus is crucial for both natural outbreaks and potential acts of bioterrorism. |
format | Online Article Text |
id | pubmed-9827366 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2004 |
publisher | United States & Canadian Academy of Pathology. |
record_format | MEDLINE/PubMed |
spelling | pubmed-98273662023-01-09 Monkeypox virus detection in rodents using real-time 3′-minor groove binder TaqMan® assays on the Roche LightCycler Kulesh, David A Loveless, Bonnie M Norwood, David Garrison, Jeffrey Whitehouse, Chris A Hartmann, Chris Mucker, Eric Miller, David Wasieloski, Leonard P Huggins, John Huhn, Gregory Miser, Lori L Imig, Carroll Martinez, Mark Larsen, Tom Rossi, Cynthia A Ludwig, George V Lab Invest Article During the summer of 2003, an outbreak of human monkeypox occurred in the Midwest region of the United States. In all, 52 rodents suspected of being infected with monkeypox virus were collected from an exotic pet dealer and from private homes. The rodents were euthanized and submitted for testing to the United States Army Medical Research Institute of Infectious Diseases by the Galesburg Animal Disease Laboratory, Illinois Department of Agriculture. The rodent tissue samples were appropriately processed and then tested by using an integrated approach involving real-time polymerase chain reaction (PCR) assays, an antigen-detection immunoassay, and virus culture. We designed and extensively tested two specific real-time PCR assays for rapidly detecting monkeypox virus DNA using the Vaccinia virus F3L and N3R genes as targets. The assays were validated against panels of orthopox viral and miscellaneous bacterial DNAs. A pan-orthopox electrochemiluminescence (ECL) assay was used to further confirm the presence of Orthopoxvirus infection of the rodents. Seven of 12 (58%) animals (seven of 52 (15%) of all animals) tested positive in both monkeypox-specific PCR assays and two additional pan-orthopox PCR assays (in at least one tissue). The ECL results showed varying degrees of agreement with PCR. One hamster and three gerbils were positive by both PCR and ECL for all tissues tested. In addition, we attempted to verify the presence of monkeypox virus by culture on multiple cell lines, by immunohistology, and by electron microscopy, with negative results. Sequencing the PCR products from the samples indicated 100% identity with monkeypox virus strain Zaire-96-I-16 (a human isolate from the Congo). These real-time PCR and ECL assays represent a significant addition to the battery of tests for the detection of various orthopoxviruses. In light of the recent monkeypox virus transmissions, early detection of the virus is crucial for both natural outbreaks and potential acts of bioterrorism. United States & Canadian Academy of Pathology. 2004-09 2023-01-04 /pmc/articles/PMC9827366/ /pubmed/15208646 http://dx.doi.org/10.1038/labinvest.3700143 Text en © 2004 United States & Canadian Academy of Pathology. Elsevier has created a Monkeypox Information Center (https://www.elsevier.com/connect/monkeypox-information-center) in response to the declared public health emergency of international concern, with free information in English on the monkeypox virus. The Monkeypox Information Center is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its monkeypox related research that is available on the Monkeypox Information Center - including this research content - immediately available in publicly funded repositories, with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the Monkeypox Information Center remains active. |
spellingShingle | Article Kulesh, David A Loveless, Bonnie M Norwood, David Garrison, Jeffrey Whitehouse, Chris A Hartmann, Chris Mucker, Eric Miller, David Wasieloski, Leonard P Huggins, John Huhn, Gregory Miser, Lori L Imig, Carroll Martinez, Mark Larsen, Tom Rossi, Cynthia A Ludwig, George V Monkeypox virus detection in rodents using real-time 3′-minor groove binder TaqMan® assays on the Roche LightCycler |
title | Monkeypox virus detection in rodents using real-time 3′-minor groove binder TaqMan® assays on the Roche LightCycler |
title_full | Monkeypox virus detection in rodents using real-time 3′-minor groove binder TaqMan® assays on the Roche LightCycler |
title_fullStr | Monkeypox virus detection in rodents using real-time 3′-minor groove binder TaqMan® assays on the Roche LightCycler |
title_full_unstemmed | Monkeypox virus detection in rodents using real-time 3′-minor groove binder TaqMan® assays on the Roche LightCycler |
title_short | Monkeypox virus detection in rodents using real-time 3′-minor groove binder TaqMan® assays on the Roche LightCycler |
title_sort | monkeypox virus detection in rodents using real-time 3′-minor groove binder taqman® assays on the roche lightcycler |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC9827366/ https://www.ncbi.nlm.nih.gov/pubmed/15208646 http://dx.doi.org/10.1038/labinvest.3700143 |
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