3.3.59
DEVELOPMENT OF LIGASE CHAIN REACTION ASSAYS FOR DETECTION OF POTATO VIRUS A and Y
DEVELOPMENT OF LIGASE CHAIN REACTION ASSAYS FOR DETECTION OF POTATO VIRUS A and Y KJ O'DONNELL, E CANNING 1 D DARLING and LGA YOUNG Scottish Agricultural Science Agency, East Craigs, Edinburgh, EHI2 8NJ Email: odonnell@sasa.gov.uk; Now at NRI, Central Avenue, Chatham Maritime, Kent Background and objectives The health of the Scottish seed potato crop is maintained by the Seed Potato Classification Scheme, which specifies strict tolerances for the level of viruses such as potato virus A (PVA) and Y (PVY) permitted in seed potato crops [11. The disease status of the growing crop is monitored by field inspections, backed up by diagnostic testing carried out by the Scottish Agricultural Science Agency (SASA). For testing of leaf material, ELISA (enzyme-linked immunosorbent assay) has been shown to provide adequate sensitivity to give accurate results. In instances where high aphid numbers have been detected late in the growing season, plants infected by aphid-borne viruses may not show symptoms until the next year. In such cases, crops are submitted for post harvest tuber testing. ELISA Is not sensitive enough to reliably detect virus In such primary infected tubers. The current test involves breaking of dormancy and planting of eyeplugs, with ELISA testing of the resulting plantlets - a process which takes 6-8 weeks. A more sensitive test would allow for more rapid turnaround of tuber samples. We have therefore investigated the possibility of using nucleic acid amplification methods, which - in theory - allow for more sensitive assays. We report here our work with one such method, the ligase chain reaction (LCR) [2J, for the detection of PVA and PVY. LCR involves two pairs of matching oligonucleotide probes. The first probes anneal to the target cDNA immediately adjacent to each other and are joined together by the action of a thermostable DNA ligase. After one heating and annealing cycle, more copies of the first pair anneal to the cDNA target and the product of the first round ligation acts as a template for the other primer pair. Repeated cycles of denaturing, annealing and ligation results In the exponential generation of ligated product from the original primers. LCR has the advantage of being easily coupled to a microtitre plate-based detection system, allowing the use of current ELISA equipment in a semi-automated assay. In the work reported here, this was done by labelling the primers with biotin or digoxigenin. This results in the generation of ligation products labelled with both, which can be easily detected in microtitre plates. LCR therefore offers the possibility of combining the sensitivity of nucleic acid-based diagnostics with the ease of use of ELI SA. Results and conclusions LCR was compared with ELISA and also with PCR. Results indicate that LCR is at least 100 times more sensitive than ELISA, using purified virus samples. Further testing using primary infected tuber samples indicates that LCR may potentially replace the existing 'growing on' method. References 1. Jeifries CJ, I 986, BCPC Monograph No.33: Healthy planting material: strategies and technologies, pp.239-247 2. O'Donnell KJ, Canning E, Young, LGA, 1996, BCPC Symposium Pmceedings No.65: Diagnostics in Crop Production, pp.187-192