Multiplex PCR for the detection of African cassava mosaic virus and East African cassava mosaic Cameroon virus in cassava
A multiplex PCR was developed for simultaneous detection of African cassava mosaic virus (ACMV) and East African cassava mosaic Cameroon virus (EACMCV) in cassava affected with cassava mosaic disease (CMD). One set of three primers consisting of an upstream primer common for both viruses and two down stream virus-specific primers were designed for simultaneous amplification of 368 base pair (bp) and 650 bp DNA fragments specific to the replicase gene of ACMV and EACMCV, respectively. Similarly, a second set of three primerswere designed for simultaneous amplification of 540 bp and 655 bp fragments specific to the coat protein gene of EACMCV and ACMV, respectively. Primers that can amplify a 171 bp fragment of the large subunit of ribulose bisphosphate carboxylase oxygenase Lwere included as an internal control in these assays to determine the reliability of multiplex PCR. A simplified, cost-effective and rapid sample preparation method was adapted in place of the conventional plant DNA extraction procedure for multiplex PCR detection of ACMV and EACMCV. The method was validated using CMD-infected cassava samples obtained from farmers’ fields in Nigeria. The multiplex PCR is useful for reliable assessment of the prevalence of CMBs in epidemiological studies and for crop improvement and phytosanitary programs in African countries.
| Main Authors: | , , |
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| Format: | Journal Article biblioteca |
| Language: | English |
| Published: |
Elsevier
2008-12
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| Subjects: | multiplex pcr, begomovirus, african cassava mosaic virus, sub saharan africa, whitefly, east african cassava mosaic cameroon virus, virology, |
| Online Access: | https://hdl.handle.net/10568/90862 https://doi.org/10.1016/j.jviromet.2008.08.008 |
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| Summary: | A multiplex PCR was developed for simultaneous detection of African cassava mosaic virus (ACMV) and
East African cassava mosaic Cameroon virus (EACMCV) in cassava affected with cassava mosaic disease
(CMD). One set of three primers consisting of an upstream primer common for both viruses and two
down stream virus-specific primers were designed for simultaneous amplification of 368 base pair (bp)
and 650 bp DNA fragments specific to the replicase gene of ACMV and EACMCV, respectively. Similarly, a
second set of three primerswere designed for simultaneous amplification of 540 bp and 655 bp fragments
specific to the coat protein gene of EACMCV and ACMV, respectively. Primers that can amplify a 171 bp
fragment of the large subunit of ribulose bisphosphate carboxylase oxygenase Lwere included as an internal
control in these assays to determine the reliability of multiplex PCR. A simplified, cost-effective and
rapid sample preparation method was adapted in place of the conventional plant DNA extraction procedure
for multiplex PCR detection of ACMV and EACMCV. The method was validated using CMD-infected
cassava samples obtained from farmers’ fields in Nigeria. The multiplex PCR is useful for reliable assessment
of the prevalence of CMBs in epidemiological studies and for crop improvement and phytosanitary
programs in African countries. |
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