Successful application of FTA® Classic Card technology and use of bacteriophage φ{symbol}29 DNA polymerase for large-scale field sampling and cloning of complete maize streak virus genomes

Betty E. Owor, Dionne N. Shepherd, Nigel J. Taylor, Richard Edema, Adérito L. Monjane, Jennifer A. Thomson, Darren P. Martin, Arvind Varsani

Research output: Contribution to journalArticle

57 Scopus citations

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 φ{symbol}29 DNA polymerase using the TempliPhi™ 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 φ{symbol}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.

Original languageEnglish (US)
Pages (from-to)100-105
Number of pages6
JournalJournal of Virological Methods
Volume140
Issue number1-2
DOIs
StatePublished - Mar 1 2007
Externally publishedYes

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Keywords

  • FTA cards
  • Geminivirus
  • MSV diversity
  • Maize streak virus (MSV)
  • Phi29 DNA polymerase
  • Rolling circle amplification

ASJC Scopus subject areas

  • Virology

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