Smallpox and
            <i>pan</i>
            -Orthopox Virus Detection by Real-Time 3′-Minor Groove Binder TaqMan Assays on the Roche LightCycler and the Cepheid Smart Cycler Platforms

Kulesh, David A.; Baker, Richard L.; Loveless, Bonnie M.; Norwood, David; Zwiers, Susan H.; Mucker, Eric M.; Hartmann, Chris; Herrera, Rafael Caballero; Miller, David; Christensen, Deanna R.; Wasieloski, Leonard P.; Huggins, John W.; Jahrling, Peter B.

doi:10.1128/jcm.42.2.601-609.2004

Cited by 127 publications

(116 citation statements)

References 33 publications

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“…19 The sensitivities of the panHA-MGB and panE9L-MGB were retested with monkeypox DNA. The LODs for both assays was 2.5 fg (12 copies).…”

Section: Animal Resultsmentioning

confidence: 99%

“…Then MPVX can be quickly and accurately distinguished from smallpox virus by the use of the MPXV-specific PCR confirmatory assays in conjunction with three previously published smallpox-specific assays. 19 …”

Section: Discussionmentioning

confidence: 99%

“…The panorthopox assays (panHA-MGB and panE9L-MGB) were previously published. 19 The MPXV assays reproducibly detected 11-55 fg of monkeypox genomic DNA, which represented approximately 50-250 copies of each gene. Results from the genomic DNA LOD experiments also correlated linearly with a dynamic range of six orders of magnitude representing approximately 25 to 2 500 000 copies (data not shown).…”

Section: Development Of Assaysmentioning

confidence: 99%

“…The first two assays, panHA-MGB and panE9L-MGB, were described previously 19 and detect the presence of orthopoxvirus DNAs. Each tissue DNA was then tested in singlet with the MPXV-specific assays, F3L-MGB and N3R-MGB.…”

Section: Assaying Of Animal Tissuesmentioning

confidence: 99%

“…[16][17][18] The goal of this study was to develop realtime LightCycler PCR assays based on the conserved F3L and N3R genes for specifically detecting MPXV and validating these assays by using a panel of DNAs from orthopox viral isolates that vary both temporally and geographically and tissues from various MPXV-infected rodents from the recent US outbreak. 8 Two previously developed pan-orthopox real-time PCR assays based on the hemagglutinin (HA) and E9L genes 19 and a pan-orthopox electrochemiluminescence (ECL) immunoassay were also used to evaluate potentially monkeypox-infected tissues.…”

mentioning

confidence: 99%

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Monkeypox virus detection in rodents using real-time 3′-minor groove binder TaqMan® assays on the Roche LightCycler

Kulesh

Loveless

Norwood

et al. 2004

Laboratory Investigation

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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 monkeypoxspecific 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.

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“…19 The sensitivities of the panHA-MGB and panE9L-MGB were retested with monkeypox DNA. The LODs for both assays was 2.5 fg (12 copies).…”

Section: Animal Resultsmentioning

confidence: 99%

Section: Discussionmentioning

confidence: 99%

Section: Development Of Assaysmentioning

confidence: 99%

Section: Assaying Of Animal Tissuesmentioning

confidence: 99%

mentioning

confidence: 99%