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 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, 826bp) 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.
International Institute of Tropical Agriculture, P. O. Box 34441, Dar es Salaam, Tanzania; University of Dar es Salaam, P.O. Box 35179, Dar es Salaam, Tanzania. Electronic address: r.shirima@cgiar.org.
Rudolph R
RR
Shirima
University of Dar es Salaam, P.O. Box 35179, Dar es Salaam, Tanzania.
Daniel G
DG
Maeda
International Institute of Tropical Agriculture, P. O. Box 34441, Dar es Salaam, Tanzania.
Edward
E
Kanju
International Institute of Tropical Agriculture, P. O. Box 34441, Dar es Salaam, Tanzania.
Gloria
G
Ceasar
Tanzania Commission for Science and Technology, P.O. Box 4302, Dar es Salaam, Tanzania.
Flora I
FI
Tibazarwa
International Institute of Tropical Agriculture, P. O. Box 34441, Dar es Salaam, Tanzania.
James P
JP
Legg
D000349:Africa; D005822:Genetic Vectors; D002365:Manihot / Q000821:virology; D019176:Potyviridae / Q000235:genetics / Q000302:isolation & purification*; D012333:RNA, Messenger / Q000032:analysis*; D012367:RNA, Viral / Q000032:analysis* / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods; D019562:Viral Load / Q000379:methods*
D012333:RNA, Messenger; D012367:RNA, Viral
Absolute quantification; CBSD; CBSV; UCBSV; Virus titre