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Development of a bacterial wilt test to facilitate the export of lucerne seed By Jan Gooden and Kathy Ophel Keller
Prepared December 2002 Published November 2005
RIRDC Publication No W05/186 RIRDC Project No SAR-10A
Antibody and DNA-based assays were considered as the basis for the Cmi seed test. The most widely used antibody assay, ELISA (enzyme-linked immunosorbent assay) is generally simple to perform, robust and internationally accepted, all important features for a routine diagnostic assay. An ELISA was developed for Cmi. The main task in developing an ELISA is to produce antibodies which detect specific proteins or polysaccharides on the target bacterium.
A rapid specific DNA assay was also developed, based on PCR (polymerase chain reaction) using primers developed by researchers in USA. The level of Cmi detected by this assay in artificially infected seed, where similar to that detected by ELISA.
The main limitation of PCR based assays is that the International Seed Testing Association (ISTA) has not supported such assays as accredited seed tests. One factor in this current view is that internationally the seed testing laboratories are not equipped to perform DNA assays.
This seems likely to change in future.
No naturally infected seed could be sourced nationally or internationally during the course of this project, so artificially spiked seed was used to develop the test.
The Cmi ELISA test developed by this project can be completed in 2 days and is relatively simple to perform. The assay is currently being evaluated as part of an international program run by the International Seed Health Initiative (SAR 25-A). Initial feedback is positive, with the assay recently detecting Cmi in a naturally infected seed lot in Canada.
A final decision by ISTA
could take several years. In the meantime the SARDI Diagnostic Centre will
begin providing the test to industry on a cost-recovery basis from 30 June
2000..
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