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 primers were 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 L were 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.
Alabi
Olufemi J
OJ
Department of Plant Pathology, Washington State University, Irrigated Agriculture Research and Extension Center, 24106 N. Bunn Road, Prosser, WA 99350, USA.
Kumar
P Lava
PL
Naidu
Rayapati A
RA
D053839:Begomovirus / Q000235:genetics / Q000302:isolation & purification*; D036022:Capsid Proteins / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D004259:DNA-Directed DNA Polymerase / Q000235:genetics; D002365:Manihot / Q000821:virology*; D010935:Plant Diseases / Q000821:virology*; D010940:Plant Proteins / Q000235:genetics; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012015:Reference Standards; D012680:Sensitivity and Specificity; D014764:Viral Proteins / Q000235:genetics
D036022:Capsid Proteins; D017931:DNA Primers; D010940:Plant Proteins; D014764:Viral Proteins; C054765:rca protein, plant; C023140:DNA replicase; D004259:DNA-Directed DNA Polymerase