Successful application of FTA® Classic Card technology and use of bacteriophage 29 DNA polymerase for large-scale field sampling and cloning of complete maize streak virus genomes
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Date
2007
Journal Title
Journal ISSN
Volume Title
Publisher
Journal of virological methods
Abstract
Leaf samples from 155 maize streak virus (MSV)-infected maize plants were collected from 155 farmers’ fields in 23 districts in Uganda in
May/June 2005 by leaf-pressing infected samples onto FTA® Classic Cards. Viral DNA was successfully extracted from cards stored at room
temperature for 9 months. The diversity of 127 MSV isolates was analysed by PCR-generated RFLPs. Six representative isolates having different
RFLP patterns and causing either severe, moderate or mild disease symptoms, were chosen for amplification from FTA cards by bacteriophage
29 DNA polymerase using the TempliPhiTM system. Full-length genomes were inserted into a cloning vector using a unique restriction enzyme
site, and sequenced. The 1.3-kb PCR product amplified directly from FTA-eluted DNA and used for RFLP analysis was also cloned and sequenced.
Comparison of cloned whole genome sequences with those of the original PCR products indicated that the correct virus genome had been cloned
and that no errors were introduced by the 29 polymerase. This is the first successful large-scale application of FTA card technology to the field,
and illustrates the ease with which large numbers of infected samples can be collected and stored for downstream molecular applications such as
diversity analysis and cloning of potentially new virus genomes.
© 2006 Elsevier B.V. All rights reserved.
Description
Keywords
Maize streak virus (MSV), Geminivirus, FTA cards, Rolling circle amplification, Phi29 DNA polymerase, MSV diversity
Citation
Owor, BE, Shepherd, DN, Taylor, NJ, Edema, R., Monjane, AL, Thomson, JA, ... & Varsani, A. (2007). Successful application of FTA® Classic Card technology and use of bacteriophage ϕ29 DNA polymerase for large-scale field sampling and cloning of complete maize streak virus genomes. Journal of virological methods , 140 (1-2), 100-105. doi:10.1016/j.jviromet.2006.11.004