DNA Banks

DNA of quarantine organisms is scarce. Therefore in this work package an investigation in different protocols to store, transport and multiply DNA samples of these organisms was made.

Objectives

  • To develop protocols for the correct storage and whole genome amplification (WGA) of extracted  DNA and RNA samples.
  • To develop a DNA bank, containing DNA/RNA and/or WGA amplified material from selected quarantine and regulated plant pathogens and their taxonomically closely related species.

Tasks

  • Task 8.1  Development protocols for correct storage of DNA samples
  • Task 8.2  Development of whole genome amplification (WGA) protocols for groups of organisms
  • Task 8.3  Development of DNA storage and transfer procedure of WGA amplified material
  • Task 8.4  Development and implementation of prototype of DNA bank
  • Task 8.5  Contingency plan

Deliverables

  • D 8.1 Two protocols for correct storage of DNA samples (Month 12)
  • D 8.2 Two protocols for WGA of DNA and RNA samples for the selected Q-organisms (Month 30)
  • D 8.3 Two protocols for the storage and transfer of WGA amplified samples for the selected Q-organisms and their relatives (Month 30)
  • D 8.4 Prototype DNA bank (Month 30)
  • D 8.5 DNA obtained by WGA for each of the 6 taxa to be used for ringtesting in WP 10 (Month 30)
  • D 8.6 Detailed contingency plan for WP8 (Month 6)

Results

DNA of quarantine organisms is scarce. Therefore in this work package an investigation in different protocols to store, transport and multiply DNA samples of these organisms was made. In this work package eight different protocols for long term storage and transport of DNA/RNA samples and WGA products were investigated and tested (e.g. filter, beads, other). GenTegra was chosen as storage medium.

Four kits for whole genome amplification (WGA), a method to multiply DNA, were tested on a subset of organisms from each group (fungi, bacteria, arthropods, nematodes, viruses, phytoplasms). The quality of the individual kits was assessed using different methods: TaqMan PCR, conventional PCR, sequence analysis and gel electrophoresis. Based upon results obtained thus far a WGA kit was selected to be used for the rest of the project.

The samples for ring testing in the work package Validation were prepared and a prototype of DNA bank was established. Several protocols have been evaluated and final protocols have been written. Using these protocols NPPO’s can better handle DNA samples of rare specimen to be used as positive and negative controls in their molecular identification and detection assays.