Development of a Multiplex TaqMan Assay for Rapid Detection of Groundnut Bud Necrosis Virus: A Quarantine Pathogen in the USA
Groundnut bud necrosis orthotospovirus (GBNV), a tripartite single-stranded RNA virus, poses a significant threat to United States agriculture. GBNV is a quarantine pathogen, and its introduction could lead to severe damage to economically important crops, such as groundnuts, tomatoes, potatoes, pea...
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| author | Anushi Suwaneththiya Deraniyagala Avijit Roy Shyam Tallury Hari Kishan Sudini Albert K. Culbreath Sudeep Bag |
| author_facet | Anushi Suwaneththiya Deraniyagala Avijit Roy Shyam Tallury Hari Kishan Sudini Albert K. Culbreath Sudeep Bag |
| author_sort | Anushi Suwaneththiya Deraniyagala |
| collection | DOAJ |
| description | Groundnut bud necrosis orthotospovirus (GBNV), a tripartite single-stranded RNA virus, poses a significant threat to United States agriculture. GBNV is a quarantine pathogen, and its introduction could lead to severe damage to economically important crops, such as groundnuts, tomatoes, potatoes, peas, and soybeans. For the rapid and accurate detection of GBNV at points of entry, TaqMan reverse transcriptase–quantitative polymerase chain reaction (RT-qPCR) assays were developed and the results validated using conventional reverse transcriptase–polymerase chain reaction (RT-PCR) followed by Sanger sequencing. These assays target highly conserved regions of the nucleocapsid (NP) and movement (MP) proteins within the viral genome. Multiplex GBNV detection assays targeting the NP and MP genes, as well as an internal control plant gene, ACT11, showed efficiency rates between 90% and 100% and R<sup>2</sup> values of 0.98 to 0.99, indicating high accuracy and precision. Moreover, there was no significant difference in sensitivity between multiplex and singleplex assays, ensuring reliable detection across various plant tissues. This rapid, sensitive, and specific diagnostic assay will provide a valuable tool at ports of entry to prevent the entry of GBNV into the United States. |
| format | Article |
| id | doaj-art-99ea8ee79f274d7cb9931f0c3e7e100b |
| institution | OA Journals |
| issn | 1999-4915 |
| language | English |
| publishDate | 2025-04-01 |
| publisher | MDPI AG |
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| series | Viruses |
| spelling | doaj-art-99ea8ee79f274d7cb9931f0c3e7e100b2025-08-20T02:18:16ZengMDPI AGViruses1999-49152025-04-0117453210.3390/v17040532Development of a Multiplex TaqMan Assay for Rapid Detection of Groundnut Bud Necrosis Virus: A Quarantine Pathogen in the USAAnushi Suwaneththiya Deraniyagala0Avijit Roy1Shyam Tallury2Hari Kishan Sudini3Albert K. Culbreath4Sudeep Bag5Department of Plant Pathology, University of Georgia, Tifton, GA 31793, USAMolecular Plant Pathology Laboratory, Beltsville Agricultural Research Center (BARC), Unites States Department of Agriculture (USDA)-Agricultural Research Service (ARS), Beltsville, MD 20705, USAPlant Genetic Resources Conservation Unit (PGRCU), United States Department of Agriculture (USDA)-Agricultural Research Service (ARS), Griffin, GA 30223, USAInternational Crops Research Institute for the Semi-Arid Tropics, Patancheru, Hyderabad 502324, Telangana, IndiaDepartment of Plant Pathology, University of Georgia, Tifton, GA 31793, USADepartment of Plant Pathology, University of Georgia, Tifton, GA 31793, USAGroundnut bud necrosis orthotospovirus (GBNV), a tripartite single-stranded RNA virus, poses a significant threat to United States agriculture. GBNV is a quarantine pathogen, and its introduction could lead to severe damage to economically important crops, such as groundnuts, tomatoes, potatoes, peas, and soybeans. For the rapid and accurate detection of GBNV at points of entry, TaqMan reverse transcriptase–quantitative polymerase chain reaction (RT-qPCR) assays were developed and the results validated using conventional reverse transcriptase–polymerase chain reaction (RT-PCR) followed by Sanger sequencing. These assays target highly conserved regions of the nucleocapsid (NP) and movement (MP) proteins within the viral genome. Multiplex GBNV detection assays targeting the NP and MP genes, as well as an internal control plant gene, ACT11, showed efficiency rates between 90% and 100% and R<sup>2</sup> values of 0.98 to 0.99, indicating high accuracy and precision. Moreover, there was no significant difference in sensitivity between multiplex and singleplex assays, ensuring reliable detection across various plant tissues. This rapid, sensitive, and specific diagnostic assay will provide a valuable tool at ports of entry to prevent the entry of GBNV into the United States.https://www.mdpi.com/1999-4915/17/4/532groundnut bud necrosis orthotospovirus (GBNV)<i>Orthotospovirus arachinecrosis</i>phytosanitary measuresTaqMan RT-qPCRquarantine pathogen |
| spellingShingle | Anushi Suwaneththiya Deraniyagala Avijit Roy Shyam Tallury Hari Kishan Sudini Albert K. Culbreath Sudeep Bag Development of a Multiplex TaqMan Assay for Rapid Detection of Groundnut Bud Necrosis Virus: A Quarantine Pathogen in the USA Viruses groundnut bud necrosis orthotospovirus (GBNV) <i>Orthotospovirus arachinecrosis</i> phytosanitary measures TaqMan RT-qPCR quarantine pathogen |
| title | Development of a Multiplex TaqMan Assay for Rapid Detection of Groundnut Bud Necrosis Virus: A Quarantine Pathogen in the USA |
| title_full | Development of a Multiplex TaqMan Assay for Rapid Detection of Groundnut Bud Necrosis Virus: A Quarantine Pathogen in the USA |
| title_fullStr | Development of a Multiplex TaqMan Assay for Rapid Detection of Groundnut Bud Necrosis Virus: A Quarantine Pathogen in the USA |
| title_full_unstemmed | Development of a Multiplex TaqMan Assay for Rapid Detection of Groundnut Bud Necrosis Virus: A Quarantine Pathogen in the USA |
| title_short | Development of a Multiplex TaqMan Assay for Rapid Detection of Groundnut Bud Necrosis Virus: A Quarantine Pathogen in the USA |
| title_sort | development of a multiplex taqman assay for rapid detection of groundnut bud necrosis virus a quarantine pathogen in the usa |
| topic | groundnut bud necrosis orthotospovirus (GBNV) <i>Orthotospovirus arachinecrosis</i> phytosanitary measures TaqMan RT-qPCR quarantine pathogen |
| url | https://www.mdpi.com/1999-4915/17/4/532 |
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