Poster by Bett BB, Kathurima TM, Miano DW, Ndolo PJ, Mwisa PN, and Kim DJ. For the BecA Opening, Nairobi, 5 November 2010.

1. Development of RT-PCR-based diagnostic technique for the
detection of Sweetpotato viruses
Bett BB1,3*, Kathurima TM2, Miano DW1, Ndolo PJ1, Mwisa PN1 and Kim DJ2
1Kenya Agricultural Research Institute, P. O. Box 57811-00200, Nairobi. 2International Institute of Tropical Agriculture
at Biosciences eastern and central Africa (BecA) Hub, ILRI, P. O. Box 30709-00100, Nairobi; 3BecA Hub.
Summary Outputs
Sweetpotato virus disease causes a 92% loss in farmers fields in Kenya. A •A diagnostic tool specific for sweetpotato viruses developed.
diagnostic test has been developed to identify the major viruses infecting •Clean and infected sweetpotato material identified.
sweetpotato. This is expected to have significant impacts in farmers’ fields.
•Ensures production of adequate healthy material for the
subsistence farmer.
Introduction
Sweetpotato is an important food security crop in Kenya grown by women mainly for household
consumption and as a source of family income. Its production however, is faced with biotic Results
constraints such as viral diseases which are the second most significant biotic constraint after the
sweetpotato weevil. The most prevalent viruses in Kenya are Sweet potato feathery mottle virus
The effectiveness of the designed primers tested on positive and negative sweetpotato
(SPFMV), Sweet potato chlorotic stunt virus (SPCSV), Sweet potato mild mottle virus control samples amplified bands as displayed in Figure 2 below. Lanes 1 to 8 are
(SPMMV), Sweet potato chlorotic fleck virus (SPCFV), and Sweet potato caulimo-like virus positive samples while the subsequent lanes 1 to 4 are negative samples (for each
(SPCaLV). Sweet potato virus disease (SPVD) is caused by the dual infection with SPFMV and primer pair) according to NCM-ELISA results.
SPCSV and is the most damaging viral disease of sweetpotato in farmers’ fields in Kenya. A
92% loss in production of three commonly grown cultivars has been reported due to the disease. B
Biotechnological interventions such as molecular-based diagnostic techniques, can ensure the A
production of virus-free planting material particularly tissue culture.
C D
A
B
Fig. 1: Diagram of A) Healthy sweetpotato planting material and B) material infected with
virus disease
Objectives
To develop a polymerase chain reaction (PCR)-based diagnostic technique to detect
sweetpotato viruses. Fig. 2: PCR gels of A): SPFMV primers A and C (185 bp); E and F (275 bp) and
Specific objectives: primer G and H (155 bp)
1.To design primers specific to sweetpotato viruses B): SPMMV primers A and B2 (268 bp); A and B (217 bp); C and F (250 bp) and
2.To evaluate their effectiveness in detecting virus infections on positive and control samples.
primer C and D1 (125 bp)
C): SPCSV primers A and B (211 bp); C and D2 (211 bp); A2 and B2 (196 bp); A2
and B (166 bp) and primer C and D (167 bp)
D): SPCFV primers A1 and B1 (135 bp); E and B1 (268 bp); C and B1 (263 bp) and
Methodology primer G and H (285 bp)
•Plant material: Sweetpotato cuttings of nine (9) farmer-preferred varieties were collected from
KARI Kakamega and serologically assayed for SPFMV, SPCSV, SPMMV, and SPCFV using
Nitro-cellulose membrane ELISA. Conclusions
•Primer design: A set of PCR Primers (forward and reverse) to detect the four viruses were •Two primer pairs were effective in testing for SPFMV and SPCFV and one
designed using the PrimerSelect software 7.0 and virus sequences deposited in the National pair for SPMMV and SPCSV.
Center for Biotechnology Information (NCBI) database. The selected virus sequences were
aligned using the Megalign software 7.0. •These primers are recommended for effective utilization in the diagnostics
•Evaluation of primers: The designed primers were tested on positive and negative samples
of tissue-cultured sweetpotato material.
obtained from the NCM-ELISA results. RNA extractions, cDNA synthesis and a PCR was
carried out using the manufacturer’s instructions obtained from the Bioneer® kit. The PCR •This will ensure the production of clean disease-free planting material for
products were ran on a 2% Agarose gel electrophoresis at 120 V for 30 minutes and viewed
under a gel documentation system.
distribution to subsistence farmers within the region.
Acknowledgement
This work was funded by the International Fund for Agricultural Research (IFAR) through the International Institute for Tropical Agriculture (IITA). Authors thank Rob Skilton, Jagger Harvey and Joel Mutisya for their critical
review and recommendation.
References
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