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Open Access Highly Accessed Methodology

Application of FTA technology for sampling, recovery and molecular characterization of viral pathogens and virus-derived transgenes from plant tissues

Joseph Ndunguru12, Nigel J Taylor1*, Jitender Yadav1, Haytham Aly3, James P Legg4, Terry Aveling2, Graham Thompson5 and Claude M Fauquet1

Author Affiliations

1 International Laboratory of Tropical Agriculture Biotechnology, Donald Danforth Plant Science Center, 975 North Warson Road, St. Louis, MO 63132, USA

2 Ministry of Agriculture and Food Security, Plant Protection Services, Box 1484, Mwanza, Tanzania

3 Department of Genetics, Faculty of Agriculture, Cairo University, Giza, Egypt

4 International Institute of Tropical Agriculture-Eastern and Southern Regional Center and Natural Resource Institute, Box 7878, Kampala, Uganda

5 ARC-Roodeplaat Vegetable and Ornamental Plant Institute, Private Bag X293, Pretoria 0001, Pretoria, South Africa

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Virology Journal 2005, 2:45  doi:10.1186/1743-422X-2-45

Published: 18 May 2005

Abstract

Background

Plant viral diseases present major constraints to crop production. Effective sampling of the viruses infecting plants is required to facilitate their molecular study and is essential for the development of crop protection and improvement programs. Retaining integrity of viral pathogens within sampled plant tissues is often a limiting factor in this process, most especially when sample sizes are large and when operating in developing counties and regions remote from laboratory facilities. FTA is a paper-based system designed to fix and store nucleic acids directly from fresh tissues pressed into the treated paper. We report here the use of FTA as an effective technology for sampling and retrieval of DNA and RNA viruses from plant tissues and their subsequent molecular analysis.

Results

DNA and RNA viruses were successfully recovered from leaf tissues of maize, cassava, tomato and tobacco pressed into FTA® Classic Cards. Viral nucleic acids eluted from FTA cards were found to be suitable for diagnostic molecular analysis by PCR-based techniques and restriction analysis, and for cloning and nucleotide sequencing in a manner equivalent to that offered by tradition isolation methods. Efficacy of the technology was demonstrated both from sampled greenhouse-grown plants and from leaf presses taken from crop plants growing in farmer's fields in East Africa. In addition, FTA technology was shown to be suitable for recovery of viral-derived transgene sequences integrated into the plant genome.

Conclusion

Results demonstrate that FTA is a practical, economical and sensitive method for sampling, storage and retrieval of viral pathogens and plant genomic sequences, when working under controlled conditions and in the field. Application of this technology has the potential to significantly increase ability to bring modern analytical techniques to bear on the viral pathogens infecting crop plants.