Absolute quantification of cassava brown streak virus mRNA by real-time qPCR
Cassava brown streak disease (CBSD) is the most important virus disease of cassava and a major food security threat in Africa. Yearly economic losses of up to $100 million USD have been attributed to CBSD. The lack of information on plant-virus interactions has restricted progress in breeding for CB...
| Main Authors: | , , , , , |
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| Format: | Journal Article |
| Language: | Inglés |
| Published: |
Elsevier
2017
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| Subjects: | |
| Online Access: | https://hdl.handle.net/10568/80816 |
| _version_ | 1855537211690188800 |
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| author | Shirima, R.R. Maeda, D.G. Kanju, E. Ceasar, G. Tibazarwa, F.I. Legg, James P. |
| author_browse | Ceasar, G. Kanju, E. Legg, James P. Maeda, D.G. Shirima, R.R. Tibazarwa, F.I. |
| author_facet | Shirima, R.R. Maeda, D.G. Kanju, E. Ceasar, G. Tibazarwa, F.I. Legg, James P. |
| author_sort | Shirima, R.R. |
| collection | Repository of Agricultural Research Outputs (CGSpace) |
| description | Cassava brown streak disease (CBSD) is the most important virus disease of cassava and a major food security threat in Africa. Yearly economic losses of up to $100 million USD have been attributed to CBSD. The lack of information on plant-virus interactions has restricted progress in breeding for CBSD resistance. Virus quantification is becoming a major tool for the quick and reliable assessment of plant host resistance. Therefore, a protocol for specific absolute quantification of Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV) was developed. CBSV and UCBSV coat protein (CP) specific standard templates: CBSV (pFer2, 826 bp) and UCBSV (pUF1-R1-1, 732) respectively were generated and maintained in a TA cloning vector. These were used to construct standard curves using a TaqMan qPCR assay. Standard curves with acceptable amplification efficiencies (90–105%) and coefficients of determination (R2) greater than 0.99 were obtained. Infected cassava plants were sampled from a screenhouse and the field and used to validate this assay. Results obtained by testing several screenhouse and field samples revealed consistent absolute quantification assays for different CBSV and UCBSV isolates. This study presents the first protocol for absolute quantification of CBSVs and is expected to accelerate screening for CBSD resistance and hence breeding for CBSD resistance. The use of the method presented here should improve the clarity of virus quantification data as the results obtained are not influenced by varietal, host, seasonal or environmental conditions. Screening efficiency will also be greatly improved as there is no need for the use of reference genes consequently allowing for a larger number of samples to be analyzed. This will increase experimental precision in a timely and cost effective manner. |
| format | Journal Article |
| id | CGSpace80816 |
| institution | CGIAR Consortium |
| language | Inglés |
| publishDate | 2017 |
| publishDateRange | 2017 |
| publishDateSort | 2017 |
| publisher | Elsevier |
| publisherStr | Elsevier |
| record_format | dspace |
| spelling | CGSpace808162025-11-11T10:43:57Z Absolute quantification of cassava brown streak virus mRNA by real-time qPCR Shirima, R.R. Maeda, D.G. Kanju, E. Ceasar, G. Tibazarwa, F.I. Legg, James P. cassava virus food sceurity dna cassava brown streak disease virus titre Cassava brown streak disease (CBSD) is the most important virus disease of cassava and a major food security threat in Africa. Yearly economic losses of up to $100 million USD have been attributed to CBSD. The lack of information on plant-virus interactions has restricted progress in breeding for CBSD resistance. Virus quantification is becoming a major tool for the quick and reliable assessment of plant host resistance. Therefore, a protocol for specific absolute quantification of Cassava brown streak virus (CBSV) and Ugandan cassava brown streak virus (UCBSV) was developed. CBSV and UCBSV coat protein (CP) specific standard templates: CBSV (pFer2, 826 bp) and UCBSV (pUF1-R1-1, 732) respectively were generated and maintained in a TA cloning vector. These were used to construct standard curves using a TaqMan qPCR assay. Standard curves with acceptable amplification efficiencies (90–105%) and coefficients of determination (R2) greater than 0.99 were obtained. Infected cassava plants were sampled from a screenhouse and the field and used to validate this assay. Results obtained by testing several screenhouse and field samples revealed consistent absolute quantification assays for different CBSV and UCBSV isolates. This study presents the first protocol for absolute quantification of CBSVs and is expected to accelerate screening for CBSD resistance and hence breeding for CBSD resistance. The use of the method presented here should improve the clarity of virus quantification data as the results obtained are not influenced by varietal, host, seasonal or environmental conditions. Screening efficiency will also be greatly improved as there is no need for the use of reference genes consequently allowing for a larger number of samples to be analyzed. This will increase experimental precision in a timely and cost effective manner. 2017-07 2017-05-02T10:26:21Z 2017-05-02T10:26:21Z Journal Article https://hdl.handle.net/10568/80816 en Open Access application/pdf Elsevier Shirima, R.R., Maeda, D.G., Kanju, E., Ceasar, G., Tibazarwa, F.I. & Legg, J. (2017). Absolute quantification of cassava brown streak virus mRNA by real-time qPCR. Journal of Virological Methods, 245, 1-13. |
| spellingShingle | cassava virus food sceurity dna cassava brown streak disease virus titre Shirima, R.R. Maeda, D.G. Kanju, E. Ceasar, G. Tibazarwa, F.I. Legg, James P. Absolute quantification of cassava brown streak virus mRNA by real-time qPCR |
| title | Absolute quantification of cassava brown streak virus mRNA by real-time qPCR |
| title_full | Absolute quantification of cassava brown streak virus mRNA by real-time qPCR |
| title_fullStr | Absolute quantification of cassava brown streak virus mRNA by real-time qPCR |
| title_full_unstemmed | Absolute quantification of cassava brown streak virus mRNA by real-time qPCR |
| title_short | Absolute quantification of cassava brown streak virus mRNA by real-time qPCR |
| title_sort | absolute quantification of cassava brown streak virus mrna by real time qpcr |
| topic | cassava virus food sceurity dna cassava brown streak disease virus titre |
| url | https://hdl.handle.net/10568/80816 |
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